Chemically defined non-polymeric valency platvorm molecules and conjugates thereof

ABSTRACT

Chemically-defined, non-polymeric valency platform molecules and conjugates comprising chemically-defined valency platform molecules and biological or chemical molecules including polynucleotide duplexes of at least 20 base pairs that have significant binding activity for human lupus anti-dsDNA autoantibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/769,041, filedDec. 19, 1996, which is a divisional of U.S. patent application Ser. No.08/453,254, filed May 30, 1995, now U.S. Pat. No. 5,606,047, which is acontinuation of U.S. patent application Ser. No. 08/152,506, filed Nov.15, 1993, now U.S. Pat. No. 5,552,391, which is a continuation-in-partof U.S. patent application Ser. No. 07/914,869 filed Jul. 15, 1992, nowU.S. Pat. No. 5,276,013; and a continuation-in-part of U.S. patentapplication Ser. No. 08/118,055, filed Sep. 8, 1993, pending. Thedisclosure of each of these parent applications is incorporated hereinby reference.

DESCRIPTION

1. Technical Field

This invention relates to conjugates comprising chemically-defined,non-polymeric valency platform molecules coupled to biological orchemical molecules such as polynucleotides for treating diseases such asthe autoimmune disease systemic lupus erythematosus (SLE or “lupus”).This invention also relates to the chemically-defined, non-polymericvalency platform molecules.

2. Background

A number of compounds have been employed as carriers for biologicallyuseful molecules in preparing conjugates that are alleged to betolerogenic. For example, Benacerraf, Katz, and their colleaguesinvestigated and described the use of conjugates of the randomco-polymer D-glutamic-acid/D-lysine, referred to as D-GL in earlierliterature (hereinafter D-EK) with haptens and various antigens toinduce specific immune tolerance. See U.S. Pats. Nos. 4,191,668 and4,220,565.

Other investigators have studied conjugates of nucleosides or DNA withother carriers. Borel et al. (Science. (1973) 182:76) evaluated theability of isogenic mouse IgG-nucleoside conjugates to reduce theantibody response to denatured DNA in young animals of the NZB mousestrain. In separate studies Parker et al. (J. Immunol. (1974) 113:292)evaluated the effect of denatured DNA conjugated, to poly-D-lysineand/or cyclophosphamide on the progression of the above-describedsyndrome in NZB mice.

In a later article (Ann NY Acad Sci (1986) 475:296-306) Borel et al.describe oligonucleotide-immunoglobulin conjugates. Borel-et al. (J ClinInvest (1988) 82:1901-1907 or U.S. Pat. No. 4,650,675) have described invitro studies using conjugates of human immunoglobulin linked to DNA.U.S. Pat. No. 5,126,131, (Dintzis et al.) also relates to conjugatescomprising carriers and molecules involved in immune responses.

Other references describe conjugates of nonimmunogenic polymers andimmunogens (Saski et al., Scand. J. Immun. (1982) 16:191-200; Sehon,Prog. Allergy (1982) 32:161-202; Wilkinson et al., J. Immunol. (987)139:326-331, and Borel et al., J. Immunol. Methods (1990) 126:159-168).

In commonly-owned U.S. Ser. No. 07/914,869, U.S. Pat. No. 5,162,515, andSer. No. 07/652,658, conjugates comprising polymeric carriers such asD-EK, polyethylene glycol, poly-D-lysine, polyvinyl alcohol, polyvinylpyrrolidone and immunoglobulins are described.

In sum, applicants believe that the prior art shows only ill-definedchemical compounds or compounds with numerous non-specific attachmentsites employed as valency platform molecules in conjugates. Because thevalency of such compounds, the specific location of the attachmentsites, and the number of attachment sites are unpredictable andfluctuates widely, prior art conjugates comprising-such compounds cannotbe made reproducibly and show wide ranges in their reported activity.

DISCLOSURE OF THE INVENTION

In contrast to the above-described art, applicants have developedconjugates comprising chemically-defined, non-polymeric valency platformmolecules wherein the valency of the platform molecules is predeterminedand wherein each attachment site is available for binding of abiological or chemical molecule. Valency platform molecules within thepresent invention are defined with respect to their chemical structure,valency, homogeneity and a defined chemistry which is amenable toeffective conjugation with the appropriate biological and/or chemicalmolecules.

Thus, one aspect of the instant invention is directed to conjugatescomprising the chemically-defined, non-polymeric valency platformmolecules and biological and/or chemical molecules. Exemplary ofbiological and/or chemical molecules suitable for conjugation tochemically-defined, non-polymeric valency platform molecules to formconjugates within the instant invention are carbohydrates, drugs,lipids, lipopolysaccharides, peptides, proteins, glycoproteins,single-stranded or double-stranded oligonucleotides and chemical analogsthereof, analogs of immunogens, haptens, mimotopes, aptamers and thelike. Chemically-defined, non-polymeric valency platform moleculessuitable for use within the present invention include, but are notlimited to, derivatives of biologically compatible and nonimmunogeniccarbon-based compounds of the following formulae: $\begin{matrix}{G^{\lbrack 1\rbrack}\left\{ T^{\lbrack 1\rbrack} \right\}_{n{\lbrack 1\rbrack}}} & {{Formula}\quad 1} \\{or} & \quad \\{G^{\lbrack 2\rbrack}\left\{ {L^{\lbrack 2\rbrack}{—J}^{\lbrack 2\rbrack}{{—Z}^{\lbrack 2\rbrack}\left( T^{\lbrack 2\rbrack} \right)}_{p{\lbrack 2\rbrack}}} \right\}_{n{\lbrack 2\rbrack}}} & {{Formula}\quad 2}\end{matrix}$wherein

-   -   each of G^([1]) and G^([2]), if present, is independently a        linear, branched or multiply-branched chain comprising 1-2000,        more preferably 1-1000, chain atoms selected from the group C,        N, O, Si, P and S;    -   more preferably, G^([2]), if present, is a radical derived from        a polyalcohol, a polyamine, or a polyglycol; most preferably,        G^([2]) is selected from the group —(CH₂)_(q)— wherein q=0 to        20, —CH₂ (CH₂OCH₂)_(r)CH₂—, wherein r=0 to 300, and        C(CH₂OCH₂CH₂—)_(s)(OH)_(4-s) wherein s=1 to 4, more preferably        s=3 to 4;    -   each of the n^([1]) moieties shown as T^([1]) and each of the        p^([2])×n^([2]) moieties shown as T^([2]) is independently        chosen from the group NHR^(SUB) (amine), C(═O)NHNHR^(SUB)        (hydrazide), NHNHR^(SUB) (hydrazine), C(═O)OH (carboxylic acid),        C(═O)OR^(ESTER) (activated ester), C(═O)OC(═O)R^(B) (anhydride),        C(═O)X (acid halide), S(═O)₂X (sulfonyl halide),        C(═NR^(SUB))OR^(SUB) (imidate ester), NCO (isocyanate), NCS        (isothiocyanate), OC(═O)X (haloformate),        C(═O)OC(═NR^(SUB))NHR^(SUB) (carbodiimide adduct), C(═O)H        (aldehyde), C(═O)R^(B) (ketone), SH (sulfhydryl or thiol), OH        (alcohol), C(═O)CH₂X (haloacetyl), R^(ALK)X (alkyl halide),        S(═O)₂OR^(ALK)X (alkyl sulfonate), NR¹R² wherein R¹R² is        —C(═O)CH═CHC(═O)— (maleimide), C(═O)CR^(B)═CR^(B) ₂        (α,β-unsaturated carbonyl), R^(ALK)—Hg—X (alkyl mercurial), and        S(═O)CR^(B)═CR^(B) ₂ (α,β-unsaturated sulfone);    -   more preferably each of the n^([1]) moieties shown as T^([1])        and each of the p^([2])×n^([2]) moieties shown as T^([2]) is        independently chosen from the group NHR^(SUB) (amine), C(═O)CH₂X        (haloacetyl), R^(ALK)X (alkyl halide), S(═O)₂OR^(ALK)X (alkyl        sulfonate), NR¹R² wherein R¹R² is —C(═O)CHCHC(═O)— (maleimide),        C(═O)CR^(B)=CR^(B) ₂ (α,β-unsaturated carbonyl), R^(ALK)—Hg—X        (alkyl mercurial), and S(═O)CR^(B)=CR^(B) ₂ (α,β-unsaturated        sulfone);    -   even more preferably each of the n^([1]) moieties shown as        T^([1]) and each of the p^([2])×n^([2]) moieties shown as        T^([2]) is independently chosen from the group NHR^(SUB)        (amine), C(═O)CH₂X (haloacetyl), NR¹R² wherein R¹R² is        —C(═O)CHCHC(═O)— (maleimide), and C(═O)CR^(B)=CR^(B) ₂        (α,β-unsaturated carbonyl);    -   most preferably, all of the n^([1]) moieties shown as T^([1])        and all of the p^([2])×n^([2]) moieties shown as T^([2]) are        identical;        wherein    -   each X is independently a halogen of atomic number greater than        16 and less than 54 or other good leaving group (i.e., weak        bases such as alkyl or alkyl-substituted sulfonates or sulfates        and the like, aryl or aryl-substituted sulfonates or sulfates        and the like that act similarly to a halogen in this setting);    -   each R^(ALK) is independently a linear, branched, or cyclic        alkyl (1-20C) group;    -   each R^(SUB) is independently H, linear, branched, or cyclic        alkyl (1-20C), aryl (6-20C), or alkaryl (7-30C);    -   each R^(ESTER) is independently N-succinimidyl, p-nitrophenyl,        pentafluorophenyl, tetrafluorophenyl, pentachlorophenyl,        2,4,5-trichlorophenyl, 2,4-dinitrophenyl, cyanomethyl and the        like, or other activating group such as 5-chloro, 8-quinolone,        1-piperidine, N-benzotriazole and the like;    -   each R^(B) is independently a radical comprising 1-50 atoms        selected from the group C, H, N, O, Si, P and S;    -   each of the n^([2]) moieties shown as L^([2]), if present, is        independently chosen from the group O, NR^(SUB) and S;    -   each of the n^([2]) moieties shown as J^([2]), if present, is        independently chosen from the group C(═O) and C(═S);    -   n^([1])=1 to 32, more preferably n^([1])=2 to 16, even more        preferably n^([1])=2 to 8, most preferably n^([1])=2 to 4;    -   n^([2])=1 to 32, more preferably n^([2])=1 to 16, even more        preferably n^([2])=1 to 8, yet more preferably n^([2])=1 to 4,        most preferably n^([2])=1 to 2;    -   p^([2])=1 to 8, more preferably p^([2])=1 to 4, most preferably        p^([2])=1 to 2;    -   with the proviso that the product n^([2])×p^([2]) be greater        than 1 and less than 33;    -   each of the n^([2]) moieties shown as Z^([2]) is independently a        radical comprising 1-200 atoms selected from the group C, H, N,        O, Si, P and S, containing attachment sites for at least p^([2])        functional groups on alkyl alkenyl, or aromatic carbon atoms;    -   more preferably, all of the n^([2]) moieties shown as Z^([2])        are identical;    -   more preferably, each of the n^([2]) moieties shown as Z^([2])        is independently described by a formula chosen from the group:        Z^([2]) is W^([3])−Y^([3]) (attachment site)_(p[2])  Formula 3        $\begin{matrix}        {Z^{\lbrack 2\rbrack}\quad{is}\quad W^{\lbrack 4\rbrack}{—N}\left\{ {Y^{\lbrack 4\rbrack}\left( {{attachment}\quad{site}} \right)}_{{p{\lbrack 2\rbrack}}/2} \right\}_{2}} & {{Formula}\quad 4} \\        {Z^{\lbrack 2\rbrack}\quad{is}\quad W^{\lbrack 5\rbrack}{—CH}\left\{ {Y^{\lbrack 5\rbrack}\left( {{attachment}\quad{site}} \right)}_{{p{\lbrack 2\rbrack}}/2} \right\}_{2}} & {{Formula}\quad 5}        \end{matrix}$        wherein    -   each of the n^([2]) moieties shown as W^([3]), W^([4]), or        W^([5]), if present, is independently a radical comprising 1-100        atoms selected from the group C, H, N, O, Si, P and S;    -   each of the n^([2]) moieties shown as Y^([3]), each of the        2×n^([2]) moieties shown as Y^([4]), and each of the 2×n^([2])        moieties shown as Y^([5]) is independently a radical comprising        1-100 atoms selected from the group C, H, N, O, Si, P and S,        containing attachment sites for at least p^([2]) (for Y^([3]))        or p^([2])/2 (for Y^([4]) and Y^([5]), where p^([2])/2 is an        integer) functional groups on alkyl, alkenyl, or aromatic carbon        atoms;    -   more preferably, each of the n^([2]) moieties shown as W^([3]),        if present, is independently chosen from the group (CH₂)_(r),        (CH₂CH₂O)_(r), NR^(SUB)(CH₂CH₂O)_(r)CH₂CH₂, and        NR^(SUB)(CH₂)_(r)NR^(SUB)C(═O), wherein r=1 to 10;    -   more preferably, each of the n^([2]) moieties shown as Y^([3])        is independently linear, branched, or cyclic alkyl (1-20C), aryl        (6-20C), or alkaryl (7-30C); most preferably, each of the        n^([2]) moieties shown as Y^([3]) is independently chosen from        the group C₆H₄, (phenyl-1,4-diradical), C₆H₃        (phenyl-1,3,5-triradical), and (CH₂)_(r) wherein r=1 to 10;    -   more preferably, each of the n^([2]) moieties shown as W^([4]),        if present, is independently chosen from the group        (CH₂)_(r)C(═O) and (CH₂)_(r)NR^(SUB)C(═O), wherein r=1 to 10;    -   more preferably, each of the 2×n^([2]) moieties shown as Y^([4])        is independently chosen from the group (CH₂)_(r),        (CH₂)_(r)NR^(SUB)C(═O) (CH₂)_(q),        (CH₂)_(r)C(═O)NR^(SUB)(CH₂)_(q), (CH₂)_(r)NR^(SUB)C(═O)        (CH₂)_(q)NR^(SUB)C(═O) (CH₂)_(r),        (CH₂)_(r)C(═O)NR^(SUB)(CH₂)_(q)NR^(SUB)C(═O) (CH₂)_(r),        (CH₂)_(r)NR^(SUB)C(═O) (CH₂CH₂O)_(q)CH₂CH₂, and        (CH₂)_(r)C(═O)NR^(SUB)(CH₂CH₂O)_(q)CH₂CH₂ wherein r=1 to 10,        more preferably r=2 to 6, and q=1 to 10, more preferably q=1 to        3;    -   more preferably, each of the n^([2]) moieties shown as W^([5]),        if present, is independently chosen from the group        (CH₂)_(r)C(═O)NR^(SUB) and (CH₂)_(r)NR^(SUB)C(═o)NR^(SUB),        wherein r=1 to 10;    -   more preferably, each of the 2×n^([2]) moieties shown as        Y^([5]), is independently chosen from the group (CH₂)_(r) and        (CH₂)_(r)C(═O)NR^(SUB)(CH₂)_(q), wherein r=1 to 10 and q=1 to        10.

In a further preferred embodiment for treating, lupus, a conjugatecomprises a chemically-defined, non-polymeric valency platform moleculeand a multiplicity of polynucleotide duplexes of at least about 20 basepairs each bound to the platform molecule, and having significantbinding activity for human SLE anti-dsDNA autoantibodies. In thesepreferred embodiments, the polynucleotide duplexes are substantiallyhomogeneous in length and one strand of the duplex is conjugated to thevalency platform molecule either directly or via a linker molecule.Usually synthetic polynucleotides are coupled to a linker moleculebefore being coupled to a valency platform molecule. Usually the linkercontaining strand of the duplex is coupled at or proximate-(i.e. withinabout 5 base pairs) one of its ends such that each strand forms apendant chain of at least about 20 base pairs measured from the site ofattachment of the strand to the linker molecule. The second strand isthen annealed to the first strand to form a duplex. Thus, a conjugatewithin the present invention can be generally described by the followingformula:[(PN)_(n)−linker]_(m)−valency platform molecule.wherein PN=a double stranded polynucleotide with “n” nucleotides,wherein n=at least about 20, and m=2-8.

Exemplary of suitable linker molecules within the present invention areCarbon thiols such as HAD, a thio-6 carbon chain phosphate, andHAD_(p)S, a thio-6 carbon chain phosphorothioate. Chemically-definedvalency platform molecules within the present invention are formed, forexample, by reacting amino modified-PEG with 3,5-bis-(iodoacetamido)benzoyl chloride (hereinafter “DABA”);3-carboxypropionamide-N,N-bis-[(6′-N′-carbobenzyloxyaminohexyl)acetamide]4″-nitrophenyl ester (hereinafter “BAHA”);3-carboxypropionamide-N,N-bis-[(8′-N′-carbobenzyloxyamino-3′,6′-dioxaoctyl)acetamide]4″-nitrophenylester (hereinafter “BAHA_(ox)”); or by reacting PEG-bis-chloroformatewith N,N-di(2-[6′-N′-carbobenzyloxyaminohexanoamido]ethyl)amine(hereinafter “AHAB”) to form chemically-defined valency platformmolecules.

Surprisingly unexpected results of at least approximately ten fold up tomore than one-hundred fold increase in immunosuppression are achievedusing conjugates comprising the chemically-defined, non-polymericvalency platform molecules of the instant invention and biological orchemical molecules (non-haptens) when compared to the polymeric carriersdescribed in the prior art. For example, at least a one hundred-foldincrease in the immunosuppression of anti-dsDNA autoantibodies wasachieved as described herein using conjugates within the presentinvention comprising chemically-defined, non-polymeric valency platformmolecules when compared to conjugates comprising an ill-defined carrierdescribed in the prior art.

Still another aspect is a conjugate of (a) a chemically-defined,non-polymeric valency platform molecule and (b) a multiplicity ofpolynucleotide duplexes each and all of which is bound to the valencyplatform molecule by a functional group located at or proximate aterminus of one of the strands of the duplex, said conjugate being ahuman SLE tolerogen.

Pharmaceutical compositions of the above-described conjugates andpharmaceutically acceptable-vehicles are another aspect of theinvention.

A further aspect of the invention is a method for treating SLE in anindividual in need of such treatment comprising administering to theindividual an effective amount of the above-described conjugates.

Yet another aspect of the invention is a method of inducing specific Bcell anergy to an immunogen in an individual comprising administering tothe individual an effective amount of the above-described conjugates.

Another aspect of the invention is a method of treating an individualfor an antibody-mediated pathology in which undesired antibodies areproduced in response to an immunogen comprising administering to theindividual an effective amount of the above-described conjugates.

A further aspect of the invention is a method for making the conjugatesdescribed-above comprising: covalently bonding the biological orchemical molecule to a chemically-defined valency platform molecule toform a conjugate.

A further aspect of the invention is a method for making the conjugatesfor treating SLE described above comprising: reacting a multiplicity ofsingle-stranded polynucleotides each of which is at least-about 20nucleotides in length and has a functional group at or proximate one ofits termini that reacts with functional groups on the chemically-definedvalency platform molecule to form a conjugate, and annealingcomplementary single-stranded polynucleotides to the single-strandedpolynucleotides conjugated to the chemically-defined valency platformmolecule to form pendant chains of double-stranded DNA.

Yet another aspect of the invention is directed to novelchemically-defined, non-polymeric valency platform molecules of theformulae: $\begin{matrix}{G^{\lbrack 6\rbrack}\left\{ {O\quad —\quad{C\left( {= O} \right)}{—NR}^{SUB}{{—Q}^{\lbrack 6\rbrack}\left( T^{\lbrack 6\rbrack} \right)}_{p{\lbrack 6\rbrack}}} \right\}_{n{\lbrack 6\rbrack}}} & {{Formula}\quad 6} \\{\quad{or}} & \quad \\{G^{\lbrack 7\rbrack}\left\{ {O\quad —\quad{C\left( {= O} \right)}{{—N}\left( {Q^{\lbrack 7\rbrack}\left( T^{\lbrack 7\rbrack} \right)}_{{p{\lbrack 7\rbrack}}/2} \right)}_{2}} \right\}_{n{\lbrack 7\rbrack}}} & {{Formula}\quad 7}\end{matrix}$wherein

-   -   each of G^([6]) and G^([7]), if present, is independently a        linear, branched or multiply-branched chain comprising 1-2000,        more preferably 1-1000, chain atoms selected from the group C,        N, O, Si, P and S; more preferably, each of G^([6]) and G^([7])        is a radical derived from a polyalcohol, a polyamine, or a        polyglycol; most preferably, each of G^([6]) and G^([7]) is        selected from the group —(CH₂)_(q)— wherein q=0 to 20,        —CH₂(CH₂OCH₂)_(r)CH₂—, wherein r=0 to 300, and        C(CH₂OCH₂CH₂—)_(s)(OH)_(4-s) wherein s=1 to 4, more preferably        s=3 to 4;    -   each of the n^([6])×p^([6]) moieties shown as T^([6]) and each        of the n^([7])×p^([7]) moieties shown as T^([7]) is        independently chosen from the group NHR^(SUB) (amine),        C(═O)NHNHR^(SUB) (hydrazide), NHNHR^(SUB) (hydrazine), C(═O)OH        (carboxylic acid), C(═O)OR^(ESTER) (activated ester),        C(═O)OC(═O)R^(B) (anhydride), C(═O)X (acid halide), S(═O)₂X        (sulfonyl halide), C(NR^(SUB))OR^(SUB) (imidate ester), NCO        (isocyanate), NCS (isothiocyanate), OC(═O)X (haloformate),        C(═O)OC(═NR^(SUB))NHR^(SUB) (carbodiimide adduct), C(═O)H        (aldehyde), C(═O)R^(B) (ketone), SH (sulfhydryl or thiol), OH        (alcohol), C(═O)CH₂X (haloacetyl), R^(ALK)X (alkyl halide),        S(═O)₂OR^(ALK)X (alkyl sulfonate), NR¹R² wherein R¹R² is        —C(═O)CHCHC(═O)— (maleimide), C(═O)CR^(B)=CR^(B) ₂        (α,β-unsaturated carbonyl), R^(ALK)—Hg—X (alkyl mercurial), and        S(═O)CR^(B)=CR^(B) ₂ (α,β-unsaturated sulfone);    -   more preferably, each of the n^([6])×p^([6]) moieties shown as        T^([6]) and each of the n^([7])×p^([7]) moieties shown as        T^([7]) is independently chosen from the group NHR^(SUB)        (amine), C(═O)CH₂X (haloacetyl), R^(ALK)X (alkyl halide),        S(═O)₂OR^(ALK)X (alkyl sulfonate), NR¹R² wherein R¹R² is        —C(═O)CHCHC(═O)— (maleimide), C(═O)CR^(B)=CR^(B) ₂        (α,β-unsaturated carbonyl), R^(ALK)—Hg—X (alkyl mercurial), and        S(═O)CR^(B)=CR^(B) ₂ (α, β-unsaturated sulfone);    -   even more preferably each of the n^([6])×p^([6]) moieties shown        as T^([6]) and each of the n^([7])×p^([7]) moieties shown as        T^([7]) is independently chosen from the group NHR^(SUB)        (amine), C(═O)CH₂X (haloacetyl), NR¹R² wherein R¹R² is        —C(═O)CH═CHC(═O)— (maleimide), and C(═O)CR^(B)=CR^(B) ₂        (α,β-unsaturated carbonyl);    -   most preferably, all of the n^([6])×p^([6]) moieties shown as        T^([6]) and all of the n^([7])×p^([7]) moieties shown as T^([7])        are identical;        wherein    -   each X is independently a halogen of atomic number greater than        16 and less than 54 or other good leaving group;    -   each R^(ALK) is independently a linear, branched, or cyclic        alkyl (1-20C) group;    -   each R^(SUB) is independently H, linear, branched, or cyclic        alkyl (1-20C), aryl (1-20C), or alkaryl (1-30C);    -   each R^(ESTER) is independently N-hydroxysuccinimidyl,        p-nitrophenoxy, pentafluorophenoxy, or other activating group;    -   each R^(B) is independently a radical comprising 1-50 atoms        selected from the group C, H, N, O, Si, P and S;    -   n^([6])=1 to 32, more preferably n^([6])=1 to 16, even more        preferably n^([6])=1 to 8, yet more preferably n^([6])=1 to 4,        most preferably n^([6])=1 to 2;    -   p^([6])=1 to 8, more preferably p^([6])=1 to 4, most preferably        p^([6])=1 to 2;    -   with the proviso that the product n^([6])×p^([6]) be greater        than 1 and less than 33;    -   n^([7])=1 to 32, more preferably n^([7])=1 to 16, even more        preferably n^([7])=1 to 8 yet more preferably n^([7])=1 to 4,        most preferably n^([7])=1 to 2,    -   p^([7])=1 to 8, more preferably p^([7])=1 to 4, most preferably        p^([7])=1 to 2;    -   with the proviso that the product n^([7])×p^([7]) be greater        than 1 and less than 33;    -   each of the n^([6]) moieties shown as Q^([6]) and each of the        2×n^([7]) moieties shown as Q^([7]) is independently a radical        comprising 1-100 atoms selected from the group C, H, N, O, Si, P        and S, containing attachment sites for at, least p^([6]) (for        Q^([6])) or p^([7])/2 (for Q^([7]), where p^([7])/2 is an        integer) functional groups on alkyl, alkenyl, or aromatic carbon        atoms;    -   more preferably, all of the n^([6]) moieties shown as Q^([6])        are identical;    -   more preferably, all of the 2×n^([7]) moieties shown as Q^([7])        are identical;    -   more preferably, each of the n^([6]) moieties shown as Q^([6]),        is independently chosen from the group CH[(CH₂)_(r)(attachment        site)]₂ and CH[(CH₂)_(r)C(═O)NR^(SUB)(CH₂)_(q)(attachment        site)]₂, wherein r=1 to 10 and q=1 to 10;    -   more preferably, each of the 2×n^([7]) moieties shown as        Q^([7]), is independently chosen from the group (CH₂)_(r),        (CH₂)_(r)NR^(SUB)C(═O) (CH₂)_(q),        (CH₂)_(r)C(═O)NR^(SUB)(CH₂)_(q), (CH₂)_(r)NR^(SUB)C(═O)        (CH₂)_(q)NR^(SUB)C(═O) (CH₂)_(r),        (CH₂)_(r)C(═O)NR^(SUB)(CH₂)_(q)NR^(SUB)C(═O) (CH₂)_(r),        (CH₂)_(r)NR^(SUB)C(═O) (CH₂CH₂O)_(q)CH₂CH₂, and        (CH₂)_(r)C(═O)NR^(SUB)(CH₂CH₂O)_(q)CH₂CH₂, wherein r=1 to 10,        more preferably r=2 to 6, and q=1 to 10, more preferably q=1 to        3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the anti-PN response in mice primed with PN-KLH, treatedwith [(PN)₂₀-BAHA]-EDDA, Conjugate 17-II, in the doses-shown, which weregiven a booster injection of PN-KLH and then bled 5 days later. Serawere tested at 3 dilutions by the Farr assay using radiolabeled PN at10⁻⁸ M and the data are presented as the percentage reduction of anti-PNantibodies. There were 5 mice per group.

FIG. 2 shows the anti-KLH response in mice primed with PN-KLH, treatedwith [(PN)₂₀-BAHA]-EDDA, Conjugate 17-II, in the doses shown, given abooster injection of PN-KLH and then bled-5 days later. Anti-KLHantibodies were assayed by enzyme-linked immunosorbent assay (ELISA).The results are expressed as the percent of a standard pool of antisera.There were 5 mice per group.

FIG. 3 shows the anti-PN response in mice primed with PN-KLH, treatedwith either [(PN)₁₆-BAHA_(OX)]-EDDA (Conjugate 11-IV),[(PN)₂₀-BAHA_(OX)]-EDDA (Conjugate 11-II), [(PN)₂₄-BAHA_(OX)]-EDDA(Conjugate 11-VI) or [(PN)₃₂-BAHA_(OX)]-EDDA (Conjugate 11-VIII) in thedoses shown, given a booster injection of PN-KLH and then bled 5 dayslater. Sera were tested by the Farr assay using radiolabeled PN at 10⁻⁸M. There were 5 mice per group.

FIG. 4 shows the anti-PN response in mice primed with PN-KLH, treatedwith (PN)₂₀-HAD-AHAB-TEG, Conjugate 20-II, in the doses shown or withHAD-AHAB only, or the PN only or a mixture of each, then boosted withPN-KLH and bled 5 days later. Sera were tested by the Farr assay usingradiolabeled PN at a concentration of 10⁻⁸ M. The percent reduction wascalculated and the data are presented. There were 5 mice per group.

FIG. 5 shows the anti-PN response in mice primed with PN-KLH, treatedwith (PN)₂₀-HAD_(p)S-AHAB-TEG, Conjugate 20-IV, in the doses shown, thenboosted with PN-KLH and, bled 5 days later. Sera were tested by the Farrassay using radiolabeled PN at a concentration of 10⁻⁸ M. There were 5mice per group.

FIGS. 6A-B show the structure of the derivatized valency platformmolecule and the linker coupling the polynucleotide to the platformmolecule for Conjugates 3-I, 3-II, 11-I, 11-II, 11-IV, 11-VI, 11-VIII,17-I, 17-II, 20-I, 20-II, 20-III, and 20-IV.

FIG. 7 shows the structures of the derivatized valency platform molecule“HAD-AHAB-TEG.”

FIG. 8 compares the level of T cell proliferation induced by melittinpeptides.

FIG. 9 compares the levels of anti-melittin peptide 2 antibodiesproduced in mice treated with melittin peptide Conjugate 2 versus thecontrol mice treated with formulation buffer.

FIG. 10 compares the levels of anti-melittin antibodies produced in micetreated with melittin peptide Conjugate 2 versus the control micetreated with formulation buffer.

FIG. 11 compares the levels of anti-melittin peptide 2 antibody-formingcells in mice treated with melittin peptide Conjugate 2 versus thecontrol mice treated with formulation buffer.

FIG. 12 illustrates that melittin peptide Conjugate 4, a conjugate ofpeptide #5 which contains a T cell epitope, was not a tolerogen.

FIG. 13 illustrates melittin conjugates within the present invention.

FIG. 14 illustrates the increase in the percentage of reduction inanti-dsPN antibody achieved by conjugates within the present inventionLJP-249A and LJP-249B which are Conjugate 3-II compared too a prior artconjugate (LJP-105) comprising D-EK and (PN)₅₀.

FIG. 15 illustrates the suppression of serum circulating IgG anti-DNAantibodies in male BXSB mice treated with LJP-394, Conjugate 20-II. AnELISA assay was used to measure IgG antibodies to (PN)₅₀ conjugated toD-EK. The serum from each of eight individual mice in each group wasassayed.

MODES FOR CARRYING OUT THE INVENTION

As used herein “valency platform molecule” means a chemically-defined,non-polymeric, nonimmunogenic molecule containing sites which facilitatethe attachment of a discreet number of biological and/or chemicalmolecules.

“Nonimmunogenic” is used to describe the valency platform molecule andmeans that the valency platform molecule elicits substantially no immuneresponse when it is administered by itself to an individual.

As used herein “individual” denotes a member of the mammalian speciesand includes humans, primates, mice and domestic animals such as cattleand sheep, sports animals such as horses, and pets such as dogs andcats.

As used herein the term “immunogen” means a chemical entity that elicitsa humoral immune response when, injected into an animal. Immunogens haveboth B cell epitopes and T cell epitopes.

The term “analog” of an immunogen intends a molecule that (a) bindsspecifically to an antibody to which the immunogen binds specificallyand (b) lacks T cell epitopes. Although the analog will normally be afragment or derivative of the immunogen and thus be of the same chemicalclass as the immunogen (e.g., the immunogen is a polypeptide and theanalog is a polypeptide), chemical similarity is not essential.Accordingly, the analog may be of a different chemical class than theimmunogen (e.g., the immunogen is a carbohydrate and the analog is apolypeptide) as long as it has the functional characteristics (a) and(b) above. The analog may be a protein, carbohydrate, lipid,lipoprotein, glycoprotein, lipopolysaccharide, nucleic acid orother-chemical or biochemical entity.

An analog of an immunogen may also comprise a “mimotope.” The term“mimotope” intends a synthetic molecule which competitively inhibits theantibody from binding the immunogen. Because it specifically binds theantibody, the mimotope is considered to mimic the antigenic determinantsof the immunogen. Like an analog of an immunogen, mimotope (a) bindsspecifically to an antibody to which the immunogen binds specificallyand (b) lacks T cell epitopes.

An analog of an immunogen may also comprise an “aptamer.” The term“aptamer” intends a synthetic oligonucleotide which competitivelyinhibits the antibody from binding the immunogen. Like an analog of animmunogen, an aptamer (a) binds specifically to an antibody to which theimmunogen binds specifically and (b) lacks T cell epitopes.

As used herein the term “B cell anergy” intends unresponsiveness ofthose B cells requiring T cell help to produce and secrete antibody andincludes, without limitation, clonal deletion of immature and/or matureB cells and/or the inability of B cells to produce antibody.“Unresponsiveness” means a therapeutically effective reduction in thehumoral response to an immunogen. Quantitatively the reduction-(asmeasured by reduction in antibody production) is at least 50%,preferably at least 75%, and most preferably 100%.

“Antibody” means those antibodies whose production is T cell dependent.

The valency of a chemically-defined valency platform molecule within thepresent invention can be predetermined by the number of branching groupsadded to the platform molecule. Suitable branching groups are typicallyderived from diamino acids, triamines, and amino diacids. A conjugatewithin the instant invention is biologically stabilized; that is, itexhibits an in vivo excretion half-life of hours to days to months toconfer therapeutic efficacy. The chemically-defined valency platformmolecules of the instant invention are also substantially nonimmunogenic(i.e., they exhibit no or only mild immunogenicity when administered toanimals), non-toxic at the doses given (i.e., they are sufficientlynon-toxic to be useful-as therapeutic agents) and are preferablycomposed of a defined chemical structure. They provide anon-immunogenic, non-toxic polyfunctional substrate to which amultiplicity of biological or chemical molecules such as polynucleotideduplexes-may be attached covalently. They will normally have an averagemolecular weight in the range of about 200 to about 200,000, usuallyabout 200 to about 20,000, and are homogeneous as compared to the priorart polymers which were a mixture of compounds of widely fluctuatingmolecular weight. Examples of particularly preferred, homogenous valencyplatform molecules within the present invention are derivatized2,2′-ethylenedioxydiethylamine (EDDA), triethylene glycol (TEG) andpolyethylene glycols (PEGs) having a molecular weight of about 200 toabout 8,000.

Conjugation of a biological or chemical molecule to thechemically-defined platform molecule may be effected in any number ofways, typically involving one or more crosslinking agents and functionalgroups on the biological or chemical molecule and valency platformmolecule.

The synthetic polynucleotide duplexes that are coupled to the valencyplatform molecule are composed of at least about 20 bp and preferably20-50 bp. Polynucleotides described herein are deoxyribonucleotidesunless otherwise indicated and are set forth in 5′ to 3′ orientation.Preferably the duplexes are substantially homogeneous in length, thatis, the variation in length in the population will not normally exceedabout +20%, preferably ±10%, of the average duplex length in base pairs.They are also preferably substantially homogeneous in nucleotidecomposition; that is, their base composition and sequence will not varyfrom duplex to duplex more than about 10%. Most preferably they areentirely homogeneous in nucleotide composition from duplex to duplex.

Based on circular dichroic (CD) spectra interpretation, the duplexesthat are useful in the invention assume a B-DNA type helical structure.It should be understood that it is not intended that the invention belimited by this belief and that the duplexes may, upon more conclusiveanalysis assume Z-DNA and/or A-DNA type helical structures.

These polynucleotide duplexes may be synthesized from native DNA orsynthesized by chemical or recombinant techniques. Naturally occurringor recombinantly produced dsDNA of longer length-may be digested (e.g.,enzymatically, chemically or by mechanical shearing) and fractionated(e.g., by agarose gel or Sephadex® column) to obtain polynucleotides ofthe desired length.

Alternatively, pairs of complementary single-stranded polynucleotidechains up to about 70 bases in length are readily prepared usingcommercially available DNA synthesizers and then annealed to formduplexes by conventional procedures. Synthetic dsDNA of longer lengthmay be obtained by enzymatic extension (5′-phosphorylation followed byligation) of the chemically produced shorter chains.

The polynucleotides may also be made by molecular cloning. For instance,polynucleotides of desired length and sequence are synthesized as above.These polynucleotides may be designed to have appropriate termini forligation into specific restriction sites. Multiple iterations of theseoligomers may be ligated in tandem to provide for multicopy replication.The resulting construct is inserted into a standard cloning vector andthe vector is introduced into a suitable microorganism/cell bytransformation. Transformants are identified by standard markers and aregrown under conditions that favor DNA replication. The polynucleotidesmay be isolated from the other DNA of the cell/microorganism bytreatment with restriction enzymes and conventional size fractionation(e.g., agarose gel, Sephadex® column).

Alternatively, the polynucleotides may be replicated by the polymerasechain reaction (PCR) technology. Saiki, R. K, et al., Science (1985)230:1350; Sacki, et al., Science (1988) 239:487; Sambrook, et al., InMolecular Cloning Techniques: A Laboratory Manual, Vol. 12, p 14.1-14.35Cold Spring Harbor Press (1989).

Polynucleotides may be screened for binding activity with SLE antiseraby the assays described in the examples. The modified Farr assay inwhich binding activity may be expressed as I₅₀ (the polynucleotideconcentration in molar nucleotides resulting in half-maximal inhibition)is a preferred assay. Polynucleotide duplexes having an I₅₀ of less thanabout 500 nM, preferably less than 50 nM, are deemed to have significantbinding activity and are, therefore, useful for making the conjugates ofthis invention.

The polynucleotides are conjugated to the chemically-defined valencyplatform molecule in a manner that preserves their antibody bindingactivity. This is done by conjugating the polynucleotide to the valencyplatform molecule at a predetermined site on the polynucleotide chainsuch that the polynucleotide forms a pendant chain of at least about 20base pairs measured from the conjugating site to the free (unattached)end of the chain.

In a particularly preferred embodiment, the polynucleotides of theinvention conjugates are coupled to a linker molecule at or proximateone of their ends. The linker molecule is then coupled to thechemically-defined valency platform molecule. For example, a defineddouble-stranded PN can-be conjugated to a valency platform molecule byfirst providing a single chain consisting of approximately 20alternating cytosine (C) and adenosine (A) nucleotides. Four CA chainscan then be covalently conjugated through linkers such as HAD to fourreactive sites on a derivatized platform molecule such as triethyleneglycol. The valency platform molecule is synthesized to include groupssuch as bromoacetyl. During the conjugation, leaving group is displacedby sulfur. A second single nucleotide chain consisting of approximately20 alternating thymidine (T) and guanosine (G) nucleotides can then beannealed to the CA strand to form a double-stranded PN conjugate of theformula, [(PN)₂₀−linker]₄−valency platform molecule.

Alternatively, in another preferred embodiment, the polynucleotide maybe coupled to the derivatized valency platform molecule at the 3′ end ofthe polynucleotide via a morpholino bridge formed by condensing anoxidized 3′ terminal ribose on one of the strands of the polynucleotidewith a free amino group on the derivatized platform molecule and thensubjecting the adduct to reducing conditions to form the morpholinolinkage. Such coupling requires the derivatized platform molecule tohave at least an equal number of amino groups as the number ofpolynucleotide duplexes to be bound to the platform molecule. Thesynthesis of such a conjugate is carried out in two steps. The firststep is coupling one strand of the polynucleotide duplex to thederivatized platform molecule via the condensation/reduction reactiondescribed above. The oxidized 3′ terminal ribose is formed on the singlepolynucleotide strand by treating the strand with periodate to convertthe 3′ terminal ribose group to an oxidized ribose group. Thesingle-stranded polynucleotide is then added slowly to an aqueoussolution of the derivatized platform molecule with a pH of about 6.0 to8.0 at 2-8° C., The molar ratio of polynucleotide to platform moleculein all the conjugation strategies will normally be in the range of about2:1 to about 30:1, usually about 2:1 to about 8:1 and preferably about4:1 to 6:1. In this regard, it is preferable that the conjugate not havean excessively large molecular weight as large molecules, particularlythose with repeating units, of m.w.>200,0000 may be T-independentimmunogens. See Dintzis et al., J. Immun. (1983) 131:2196 and J. Immun.(1989) 143:1239. During or after the condensation reaction (normally areaction time of 24 to 48 hr), a strong reducing agent, such as sodiumcyanoborohydride, is added to form the morpholino group. Thecomplementary-strand of the duplex is then added to the conjugate andthe mixture is heated and slowly cooled to cause the strands to anneal.The conjugate may be purified by gel permeation chromatography.

An alternative to the ribose strategy is forming aldehydefunctionalities on the polynucleotides and using those functionalitiesto couple the polynucleotide to the platform molecule via reactivefunctional groups thereon. Advantage may be taken of the fact that gem,vicinal diols, attached to the 3′ or 5′ end of the polynucleotide, maybe oxidized with sodium periodate to yield aldehydes which can condensewith functional amino groups of the platform molecule. When the diolsare in a ring system, e.g., a five-membered ring, the resultingcondensation product is a heterocyclic ring containing nitrogen, e.g., asix-membered morpholino or piperidino ring. The imino-condensationproduct is stabilized by reduction with a suitable reducing agent; e.g.,sodium borohydride or sodium cyanoborohydride. When the diol is acyclic,the resulting oxidation product contains just one aldehyde and thecondensation product is a secondary amine.

Another procedure involves introducing alkylamino or alkylsulfhydrylmoieties into either the 3′ or 5′ ends of the polynucleotide byappropriate nucleotide chemistry, e.g., phosphoramidate chemistry. Thenucleophilic groups may then be used to react with a large excess ofhomobifunctional cross-linking reagent, e.g., dimethyl suberimidate, inthe case of alkylamine derivatives, or an excess of heterobifunctionalcross-linking reagent, e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester (MBS) or succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), for thealkylsulfhydryl derivatives. Once excess cross-linker is removed, thepolynucleotide derivatives are reacted with amino groups on the platformmolecule. Alternatively; the sulfhydryl group may be reacted with anelectrophilic center on the platform, such as a maleimide orα-haloacetyl group or other appropriate Michael acceptor.

Still another strategy employs modified nucleosides. Suitabledeoxynucleoside derivatives can be incorporated, by standard DNAsynthetic chemistry, at desired positions in the polynucleotide,preferably on the 5′ or 3′ ends. These nucleoside derivatives may thenreact specifically and directly with alkylamino groups on the platformmolecule. Alternatively, side reactions seen with the above-describeddialdehyde chemistry, such as amine catalyzed beta-elimination, can becircumvented by employing appropriate nucleoside derivatives as the 3′terminus of the chain to be attached. An example of this is 5′ methyleneextension of ribose; i.e., a 5′ (2-hydroxyethyl)-group instead of a 5′hydroxymethyl group. An alternative would be to use a phosphonate orphosphinate linkage for the 3′ terminal dinucleotide of thepolynucleotide to be attached to the platform molecule.

Analogs of Immunogens

Immunogens that are involved in antibody-mediated pathologies may beexternal (foreign to the individual) immunogens such as allergens,α-sperm associated with male infertility, the rheumatic fevercarbohydrate complex, the RBC Rh/D antigen associated with hemolyticdisease of the newborn, biological drugs, including native biologicalsubstances foreign to the individual such as therapeutic proteins,peptides and antibodies, and the like or self-immunogens(autoimmunogens) such as those associated with thyroiditis(thyroglobulin), stroke (cardiolipin) and myasthenia gravis(acetylcholine receptor).

Analogs to such immunogens may be identified by screening candidatemolecules to determine whether they (a) bind specifically to serumantibodies to the immunogen and (b) lack T cell epitopes. Specificbinding to serum antibodies may be determined using conventionalimmunoassays and the presence or absence of T cell epitopes may bedetermined by conventional T cell activation assays. In this regard, ananalog which “binds specifically” to serum antibodies to the immunogenexhibits a reasonable affinity thereto. Further in this regard, itshould be recognized that testing for T cell epitopes is conducted on asubject-by-subject basis using T cells taken from an intended recipientor from various patients that represent the target population ofrecipients. The presence or absence of T cell epitopes may be determinedusing the tritiated thymidine incorporation assay described in theexamples. The presence of T cell eptiopes can also be determined bymeasuring secretion of T cell-derived lymphokines by methods well knownin the art. Analogs that fail to induce statistically significantincorporation of thymidine above background are deemed to lack T cellepitopes. It will be appreciated that the quantitative amount ofthymidine incorporation may vary with the immunogen. Typically astimulation index below about 2-3, more usually about 1-2, is indicativeof a lack of T cell epitopes.

A normal first step in identifying useful analogs is to prepare a panelor library of candidates to screen. For instance, in the case ofprotein-or peptide analogs, libraries may be made by synthetic orrecombinant techniques such as those described by Geysen et al. inSynthetic Peptides as Antigens; Ciba Symposium (1986) 119:131-149;Devlin et al., Science (1990) 249:404-406; Scott et al., Science (1990)249:386-390; and Cwirla et al., PNAS USA (1990) 87:6378-6382. In onesynthetic technique, peptides of about 5 to 30 amino acids aresynthesized in such a manner that each peptide overlaps the next and alllinear epitopes are represented. This is accomplished by overlappingboth the carboxyl and amino termini by one less residue than thatexpected for a B cell epitope. For example, if the assumed minimumrequirement for a B cell epitope is six amino acids, then each peptidemust overlap the neighboring peptides by five amino acids. In thisembodiment, each peptide is then screened against antisera producedagainst the native immunogen, either by immunization of animals or frompatients, to identify the presence of B cell epitopes. Those moleculeswith antibody binding activity, are then screened for the presence of Tcell epitopes as described in the examples. The molecules lacking T cellepitopes are useful as analogs in the invention.

If the T cell epitope(s) of an immunogen are known or can be identified,random T cell screening of candidate analogs is not necessary. In suchinstances, the T cell epitope(s) may be altered (e.g., by chemicalderivatization, or elimination of one or more components of the epitope)to render them inoperative or be eliminated completely, such as, forinstance, in the case of peptides, by synthetic or recombinantprocedures.

Mimotopes and aptamers are synthesized by conventional methods and arescreened in the same manner as other analogs of immunogens.

The analogs are coupled to a nonimmunogenic valency platform molecule toprepare the conjugates of the invention. Conjugates comprising valencyplatform molecules and biologically active molecules such ascarbohydrates, lipids, lipopolysaccharides, proteins, glycoproteins,drugs, and analogs of interest are synthesized utilizing the chemistriesexemplified herein. A preferred method of synthesis is to incorporate alinker molecule on the biological molecule by well known methods chosenon a case-by-case basis.

When conjugating drugs such as adriamycin, (doxorubicin) to a valencyplatform molecule, the amino group on a sugar ring can react withplatform molecules containing active esters. Adriamycin can also bemodified to contain thiol groups for conjugation to a haloacetylatedplatform (Kaneko, T., et al., Bioconjugate Chemistry, 2:133 (1991)).

Carbohydrates such as oligosaccharides can be modified to contain asulfhydryl-containing linker (Wood, S. J. and Wetzel, R., BioconjugateChemistry, 3:391 (1992)). The sulfhydryl group is used for conjugationto a haloacetylated platform. Alternatively, carbohydrates can beoxidized to generate aldehydes which is reacted in the presence ofNaCNBH₃ with amino platforms to form conjugates.

Lipids such as glycol-lipids containing an ethanolamine group arereacted with an activated carboxylate on the platform.Lipopolysaccharides containing sugar units are oxidized to generatealdehydes which are reacted in the presence of NaCNBH₃ with aminoplatforms to form conjugates by reductive amination.

In the case of additional proteins such as Fab′ antibody fragments,sulfhydryl groups on the protein (Fab′) are conjugated to a platform viahaloacetyl groups. Glycoproteins are modified with a thiol linker usingiminothiolate. The thiol reacts with platforms containing haloacetyl,groups.

The ability of the conjugates to act as tolerogens and specificallysuppress production of antibodies may be evaluated in the murine modeldescribed in the examples.

The conjugates will normally be formulated for administration byinjection, (e.g., intraperitoneally, intramuscularly, intravenouslyetc.). Accordingly, they will typically be combined withpharmaceutically acceptable aqueous carriers such as saline, Ringer'ssolution, dextrose solution, and the like. The conjugate will normallyconstitute about 0.01%- to 10% by weight of the formulation. Theconjugate is administered to an individual in amounts sufficient to atleast partially reestablish tolerance to the autoantigens causing SLE.Such amounts are sometimes herein referred to as “therapeuticallyeffective” amounts. The particular dosage regimen i.e., dose, timing andrepetition, will depend upon the particular individual, and thatindividuals medical history. Normally a dose of about 1 to 1000 μgconjugate/kg body weight will be given. Repetitive administrations maybe required to achieve and/or maintain a state of immune tolerance.

The following examples further illustrate the invention and itsunexpectedness relative to the prior art. These examples are notintended to limit the invention in any manner.

EXAMPLE 1

The following reaction schemes illustrate methods of synthesizingderivatized chemically-defined valency platform molecules within thepresent invention. In this example, DMTr=dimethoxytrityl; Tr=trityl;Bz=benzoyl; Cp=deoxycytidine monophosphate, CE=cyanoethyl;CPG=controlled pore glass, DMF=dimethyl formamide,DCC=dicyclohexylcarbodiimide, TFA=trifluoroacetic acid, CDI=carbonyldiimidazole, Ts=tosyl (para-toluene sulfonyl), DIPAT=diisopropylammonium tetraazolide, TBDMSCl=tetrabutyl dimethyl silyl chloride,TBAF=tetrabutyl ammonium fluoride, NMMO=N-methylmorpholine oxide.

Synthesis of reagents used to modify (CA)₈,(CA)₁₀, (CA)₁₂ and (CA)₁₆with disulfide linkers is described in Reaction Scheme 11 below:

Synthesis of a reagent used to modify (CA)₂₅ with vicinal diol linkersis described in Reaction Scheme 12 below:

EXAMPLE 2 Synthesis of Chemically-Defined Valency Platform Molecules

Compound 1-[3,5-Bis-(iodoacetamido)benzoic acid]: 2.93 g (8.28 mmol, 2.2eq) of iodoacetic anhydride was added to a stirred suspension of 572 mg(3.76 mmol) of 3,5-diaminobenzoic acid in 19 mL of dioxane at roomtemperature under N₂ atmosphere. The mixture was stirred, covered withfoil for 20 hours and partitioned between 50 mL of EtOAc and 50 mL of 1NHCl solution. The EtOAc layer was washed with brine, dried over MgSO₄,filtered, and concentrated on a rotary evaporator to give 3.3 g of tansolid. The material was purified by silica gel chromatography (94/5/1CH₂Cl₂/MeOH/HOAc) to yield 992 mg (54%) of compound 1 as a white solid:NMR (DMSO) 3.84 (s, 4H), 7.91 (s, 2H), 8.14 (s, 1H), 10.56 (s, 2H).

Compound 2-[3,5-Bis-(iodoacetamido)benzoyl chloride]: 117 μL (1.6 mmol,190 mg) of SOCl₂ was added to a solution of 390 mg (0.8 mmol) of 1 in 34mL of THF. The mixture was refluxed under N₂ atmosphere until all solidshad dissolved (approximately 30 minutes) to give a clear red-brownsolution. The mixture was concentrated on the rotary evaporator andplaced under vacuum to provide crude compound 2 as a foamy solid whichwas used directly in the next step.

Compound 3-[N,N′-Bis-(3,5-bis-(iodoacetamido)benzoyl) derivative ofα,ω-bis-(N-2-aminoethylcarbamoyl)polyethyleneglycol]: 570 mg ofα,ω-bis-(N-2-aminoethylcarbamoyl)polyethyleneglycol (0.16 mmol, 3350g/mol, Sigma) was placed in a tared flask. Toluene (20 mL) was added andwater was removed by azeotropic distillation. The residue was driedunder vacuum to give 549 mg of solid and dissolved in 4 mL THF with 89μL (0.64 mmol) of diisopropylethylamine. The crude acid chloride wasdissolved in 4 mL anhydrous THF and added to the mixture over 30 secondsunder N₂. The mixture was stirred for 16 hours at room temperature andpartitioned between 25 mL of 0.1 N HCl and 25 mL of CH₂Cl₂. The aqueouslayer was again extracted with CH₂Cl₂ and the organic layers werecombined, washed with 25 mL of H₂O, followed by 50 mL of at NaHCO₃solution. The organic layers were dried with Na₂SO₄, filtered, andconcentrated to give 784 mg of orange oil. Silica gel chromatography(9/1 CH₂Cl₂/MeOH) yielded 190 mg of colorless oil which was-crystallizedfrom hot EtOH/Et₂O, collected on sintered glass, filter under N₂pressure, and dried under vacuum to provide 177 mg of compound 3 as awhite solid: NMR (CDCl₃) 3.40 (bd m, 8H), 3.59 (bd s, (CH₂CH₂O)_(n),integral too large to integrate in relation to other-integrals), 3.91(s, 8H), 4.21 (m, 4H), 6.04 (bd m, 2H), 7.55 (bd m, 2H), 7.78 (bd s,4H), 8.10 (bd s, 2H), 9.30 (bd m, 4H): iodoacetyl determination(European Journal of Biochemistry (1984) 140:63-71): Calculated, 0.92mmol/g; Found, 0.96 mmol/g.

Compound 4-[Mono-N-carbobenzyloxy-3,6-dioxa-1,8-diaminooctane]: Asolution of 14.3 mL (17.1 g, 100 mmol) of benzylchloroformate in 200 mLof CH₂Cl₂ was added dropwise over a 1 hour period to a solution of 29.0mL (29.6 g, 200 mmol) of 2,2′-(ethylenedioxy)-diethylamine (Fluka) in100 mL of CH₂Cl₂ at 0°. The mixture was stirred at room temperature for24 hours and 1 N HCl was added until the aqueous layer remained acidic(pH less than 2). The aqueous layer was washed with three 50 mL portionsof CH₂Cl₂ and neutralized with 1 N NaOH until the pH was above 13. Thebasic aqueous layer was extracted with five 75 mL portions of CH₂Cl₂.The combined CH₂Cl₂ layers were dried (MgSO₄), filtered, andconcentrated to yield 12.7 g (45%) of compound 4 as a thick oil: ¹H NMR(CDCl₃) d 2.82 (bd s, 2H), 3.30-3.60 (m, 12H), 5.10 (s, 2H), 5.75 (bd s,1H), 7.20-7.40 (m, 5H); ¹³C NMR (CDCl₃) d 41.1, 41.8, 66.5, 70.0, 70.2,70.4, 73.5, 127.9, 128.0, 128.4, 136.9, 156.4.

Compound 5-[N-tert-butyloxycarbonyliminodiacetic acid]: This compoundwas prepared by a procedure similar to that reported by Garrigues, B.and Lazraq, E. M. Tetrahedron Letters (1986) 27, 1685-1686. 47 mL (34.2g, 338 mmol) of Et₃N was added to a stirred solution of 22.0 g (169mmol) of iminodiacetic acid and 36.8 g (169 mmol) of di-tertbutyldicarbonate in 169 mL of 50/50 dioxane/H₂O at room temperature. Themixture was stirred for 24 hours and most of the dioxane was removed ona rotary evaporator. The mixture was partitioned between 350 mL of 1 NHCl and five 100 mL portions of EtOAc. The combined EtOAc layers weredried (MgSO₄), filtered, and concentrated to give a white solid.Recrystallization from hexanes/EtOAc yielded 35.3 g (90%) of compound 5as crystals: m.p. 131-132° fused; ¹H NMR (DMSO) d 1.35 (s, 9H), 3.87 (s,2H), 3.91 (s, 2H), 12.6 (bd s, 2H); ¹³C NMR (DMSO) d 27.9, 49.6, 79.6,154.8, 171.2.

Compound 6. 9.99 g (48.5 mmol) of dicyclohexylcarbodiimide was added toa solution of 4.52 g 73 (19.4 mmol) of compound 5 and 4.46 g (38.8 mmol)of N-hydroxysuccinimide in 100, mL of THF at 0°. Th mixture was stirredfor 3 hours at 0° C., and a solution of 5.39 mL (3.92 g, 38.8 mmol) Et₃Nand 10.9 g (38.7 mmol) of compound 4 in 83 mL of THF was added, and themixture was stirred at 5° C. for 17 hours. The mixture was filtered toremove solids, and the filtrate was concentrated to an oil which waspartitioned between 400 mL of EtOAc and two 100 mL portions of 1 N HCl.The EtOAc layer was washed with three, 100 mL portions of 1 N Na₂CO₃,100 mL of brine, dried (MgSO₄), filtered and concentrated to provide14.2 g (96%) of compound 6 as a thick oil; ¹H NMR (CDCl₃) d 1.41 (s,9H), 3.30-3.70 (m, 24H), 3.70-3.90 (m, 4H), 5.10 (s, 4H), 5.50 (bd s,2H), 7.12 (bd s, 1H), 7.30-7.40 (m, 10H), 8.24 (bd s, 1H).

Compound 7. 26.3 mL (38.9 g, 156 mmol) of trifluoroacetic acid was addedtoga solution of 14.2 g (18.6 mmol) of compound 6 in 111 mL of CH₂Cl₂and the mixture was stirred at room temperature for 3 hours. The mixturewas concentrated on the rotary evaporator to give a viscous oil, and theoil was dissolved in 93 mL of THF. The solution was cooled to 0° C. and3.72 g (37.2 mmol) of succinic anhydride was added followed by 5.18 mL(3.76 g, 37.2 mmol) of Et₃N. The cooling bath was removed, and themixture was stirred for 18 hours at room temperature. The solvent wasremoved under reduced pressure, and the resulting oil was partitionedbetween 300 mL of CH₂Cl₂ and three 100 mL portions of H₂O. The CH₂Cl₂layer was dried (MgSO₄), filtered, and concentrated to provide an oilwhich was purified by chromatography on silica gel (9/1/0.1EtOAc/MeOH/acetic acid) to provide 10.5 g (74%) of compound 7 as aviscous oil; ¹H NMR (CDCl) d 2.50-2.60 (m, 4H), 3.30-3.60 (m, 24H), 3.88(s, 2H), 4.03 (s, 2H), 5.07 (s, 4H), 5.77 (bd s, 2H), 7.20-7.30 (m 10H),7.91 (bd s, 2H), 8.88 (bd s, 1H); ¹³C (CDCl₃) d 27.7, 29.0, 39.4, 41.0,52.9, 53.8, 66.5, 69.3, 69.8, 70.0, 70.1, 127.8, 128.1, 128.3, 136.7,156.6, 169.1, 169.6, 173.3, 174.5.

Compound 8-[4-Nitrophenyl ester of compound 7]: 1.61 g (7.83 mmol) ofdicyclohexylcarbodiimide was added to a solution of 3.98 g (5.22 mmol)of 7 and 800 mg (5.75 mmol) of 4-nitrophenol in 26 mL of CH₂Cl₂ at 0°.The mixture was stirred at room temperature under N₂ for 64 hours. Themixture was cooled to 0°, 1 mL of HOAc was added, and the mixture waskept at 0°0 for 2 hours. The solids were removed by filtration, and thefiltrate was concentrated. The residue was purified by silica gelchromatography, (gradient, 91/8/1 to 84/15/1 CH₂Cl₂/IPA/HOAc) to provide2.58 g (56%) of compound 8 as a viscous oil: ¹H NMR (CDCl₃) d 2.66 (t,2H), 2.84 (t, 2H), 3.32-3.68 (m, 24H), 3.90 (bd s, 2H), 4.01 (bd s, 2H),5.06 (s, 4H), 5.58 (bd m, 2H), 6.91 (bd m, 1H), 7.27 (d, 2H), 7.33 (s,10H), 8.23 (d, 2H), 9.01 (bd m, 1H).

Compound 10-[4-Nitrophenylbromoacetate]: 9.28 g (45 viol) ofdicyclohexylcarbodiimide was added to a stirred solution of 5.0 g (35.9mmol) of bromoacetic acid and 8.50 g (61.1 mmol) of 4-nitrophenol in 180mL of EtOAc at 0°. The mixture was stirred for 16 hours at 5° and 1 mLof acetic acid was added. The mixture was stirred for 20 minutes at roomtemperature and then placed in a freezer for 20 minutes. The solidmaterial was removed by filtration, and the filtrate was concentrated toa viscous oil and crystallized from Et₂O/hexanes to provide 7.73 g (83%)of compound 10 as flak s: m.p. 86-87°; TLC Rf=0.63 (50/50/1hexanes/EtOAc/HOAc); ¹H NMR (CDCl₃) d 4.13 (s, 2H), 7.36 (d, J=12 Hz,2H), 8.32 (d, J=12 Hz, 2H); ¹³C NMR (CDCl₃) d 24.9, 122.1, 125.3, 155.5164.9; Anal. calc'd for C₈H₆BrNO₄: C, 36.95; H, 2.33; N, 5.39. Found: C,37.24; H, 2.33; N, 5.42.

Compound 9: 300 mg (3.57 mmol) of NaHCO₃, followed by 162 mg (1.09 mmol)of 2,2₁-(ethylenedioxy)-diethylamine (Fluka), was added to a solution of2.37 g (2.68 mmol) of compound 8 in 15 mL of dioxane and 8 mL of H₂O.The mixture was stirred for 24 hours at room temperature andconcentrated under vacuum to approximately one half the original volume.The concentrate was partitioned between 40 mL of CH₂Cl₂ and 40 mL ofsaturated NaHCO₃ solution. The CH₂Cl₂ layer was then washed twice with40 mL of 0.5 N HCl. The CH₂Cl₂ layer was washed with saturated NaClsolution, dried (MgSO₄), filtered, and concentrated to give 2.8 g of anoil. This crude produce was purified by silica gel chromatography (3/6/1CH₂Cl₂/THF/MeOH) to provide 940 mg (59%) of compound 9 as an oil: TLCR_(f)=0.21 (3/6/1 CH₂Cl₂/THF/MeOH); ¹H NMR (CDCl₃) d 2.45 (m, 4H), 2.59(m, 4H), 3.25-3.55 (m, 60H), 3.87 (s, 4H), 4.05 (s, 4H), 5.07 (s, 8H),5.62 (bd s, 4H), 6.78 (bd s, 2), 7.34 (bd s, 20H), 8.56 (bd s, 2H); ¹³CNMR (CDCl₃) d 28.1, 30.3, 31.1, 39.4, 41.1, 52.9, 53.9, 66.5, 69.4,69.7, 69.9, 70.2, 125.3, 127.8, 128.3, 136.8, 156.5, 168.8, 169.4,172.1, 173.5.

Compound 34: 110 mg of 10% Pd on carbon was added to a solution of 281mg (0.175 mmol) of compound 9 in 5 mL of EtOH and 2 mL of cyclohexeneunder nitrogen and the resulting mixture was refluxed under nitrogen for2 hours. When cool, the mixture was filtered through diatomaceous earthand concentrated under vacuum to give 170 mg (92%) of compound 34 as anoil which was used directly in the next step without purification; ¹HNMR (CDCl₃) d 2.45 (m, 4H), 2.53 (m, 4H), 2.62 (m, 4H), 2.86 (m, 8H),3.42-3.60 (m, 52H), 4.00 (s, 4H), 4.14 (s, 4H); ¹³C NMR (CDCl₃) d 28.2,30.3, 31.1, 39.4, 41.1, 46.5, 48.6, 52.9, 53.8, 69.4, 69.7, 70.2, 72.4,168.9, 169.5, 172.3, 173.8.

Compound 11: 128 mg (1.4 mmol) of NaHCO₃ and 200 mg (0.85 mmol) ofcompound 10 were added to a solution of 165 mg (0.155 mmol) of compound34 in 6 mL of dioxane and 3 mL of H₂O. The resulting mixture was stirredfor 24 hours at room temperature and concentrated under vacuum. Theconcentrate was purified by chromatography on Sephadex® G-10 (MeOH) togive 114 mg (46%) of compound 11 as a viscous oil. An analytical samplewas prepared by preparative HPLC (C₁₈; gradient 15/85/0.1 to30/70/0.1CH₃CN/H₂O/CF₃CO₂H, 50 min, 225 nm): ¹H NMR (CDCl₃) d 2.58 (m,4H), 2.65 (m, 4H), 3.43-3.62 (m, 60H), 3.92, (s, 8H), 4.03 (s, 4H), 4.16(s, 4H); MS (FAB) m/e (relative intensity) MNa+ 1605 (100), MH+ 1579(1), 1581 (5), 1583 (7), 1585 (6), 1587 (2).

Compound 12-[Mono-N-carbobenzyloxy-1,6-diaminohexane]: A solution of 21mL (25.7 g, 150 mmol) of benzylchloroformate in 200 mL of dioxane wasadded dropwise to a stirred solution of 17.49 g (150 mmol) of1,6-hexanediamine and 19.58 g (196 mmol) of KHCO₃ in 100 mL of dioxaneand 300 mL of H₂O at 0°. The mixture was stirred at room temperature for18 hours and then cooled to 0°. The mixture was acidified with 12 N HCland extracted with two 100 mL portions of Et₂O. The aqueous layer wasneutralized with 10 N NaOH and extracted with eight 100 mL portions ofEt₂O. The basic extracts were combined, dried (Na₂SO₄), and concentratedto provide 5.03 g (13%) of crude compound 12 as a semisolid residue: ¹HNMR (DMSO) d 1.22-1.51 (m, 8H), 2.54 (t, 2H), 3.02 (d of t, 2H), 5.05(s, 2H), 7.30-7.48 (m, 5H).

Compound 13: 918 mg (4.45 mmol) of dicyclohexylcarbodiimide was added toa solution of 417 mg (1.78 mmol) of compound 5 and 409 mg (3.56 mmol) ofNHS in 15 mL of THF at 0°. The mixture was stirred at 0° for 4.5 hoursand a solution of 1.02 g (4.08 mmol) of compound 1 in 4 mL of THF wasadded. The mixture was stirred under N₂ at 5° for 18 hours. Theconcentrate was partitioned between 30 mL of EtOAc and two 30 mLportions of 1 N HCl. The combined EtOAc layers were washed successivelywith 30 mL of H₂O and 30 mL of saturated NaHCO₃ solution, dried (MgSO₄),filtered, and concentrated to provide 1.48 g of viscous residue.Purification by chromatography on silica gel (5/95 MeOH/CH₂Cl₂) gave1.04 g (84%) of, compound 13 as a sticky solid: ¹H NMR (CDCl₃) d 1.33(m, 8H), 1.43 (s, 9H), 1.51 (m, 8H), 3.18 (m, 4H), 3.26 (m, 4H), 3.81(s, 2H), 3.85 (s, 2H), 4.90 (bd s, 2H), 5.10 (s, 4H), 6.81 (bd s, 1H),7.28-7.40 (m, 10H), 8.05 (bd s, 1H).

Compound 14: 14.9 mL of trifluoroacetic acid was added to a solution of5.16 g (7.45 mmol) of compound 13 in 14.9 mL of CH₂Cl₂ and the resultingmixture was stirred for 3 hours at room temperature. The mixture wasconcentrated under vacuum and redissolved in 57 mL of THF. 2.07 mL (1.51g, 14.9 mmol) of Et₃N was added to the mixture. 1.5 g (14.9 mmol) ofsuccinic anhydride was added to the mixture and the mixture was thenstirred for 18 hours. The mixture was partitioned between 75 mL of 1 NHCl and four 75 mL portions of CH₂Cl₂. The combined CH₂Cl₂ layers weredried (MgSO₄), filtered, and concentrated to provide a solid.Crystallization from CH₂Cl₂/EtOAc/hexanes provided 3.84 g (74%) ofcompound JA as a white solid: m.p. 1220; ¹H NMR (MeoH) d 1.32 (m, 8H),1.48 (m, 8H), 2.56 (m, 4H), 3.10 (t, 4H), 3.23 (m, 4H), 4.00 (s, 2H),4.18 (s, 2H), 5.05 (s, 4H), 7.33 (m, 10H).

Compound 15-[4-Nitrophenyl ester of compound 14]: 887 mg (4.30 mmol) ofdicyclohexylcarbodiimideo was added to a solution of 2.0 g (2.87 mmol)of compound 14 and 438 mg (3.15 mmol) of 4-nitrophenol in 15 mL of THFat 0°. The mixture was allowed to come to room temperature, stirred for18 hours, and then cooled to 0°. 200 uL of acetic acid was then addedand the mixture was stirred at 0° for 1 hour. The solids were removed byfiltration and the filtrate was concentrated to an oil. Purification bychromatography on silica gel (92/8 CH₂Cl₂/IPA) and recrystallization ofthe resulting solid from CH₂Cl₂/hexanes provided 1.52 g (64%) ofcompound 15 as a white solid: m.p. 65-68°; ¹H NMR (CDCl₃) d, 1.30 (m,5H), 1.47 (m, 8H), 2.71 (t, 2H), 2.90 (t, 2H), 3.17 (m, 4H), 3.25 (m,4H), 3.92 (s, 2H), 4.08 (s, 2H), 4.86 (bd t, 1H), 4.95 (bd t, 1H), 5.09(s, 4H), 6.28 (bd t, 1H), 7.23 (d, J=9 Hz, 2H), 7.32 (m, 10H), 8.22 (d,J=9 Hz, 2H), 8.95 (bd t, 1H).

Compound 16: A solution of 830 mg (0.99 mmol) of compound 15 in 7.5 mLof dioxane was added to a solution of 58 uL (59 mg, 0.40 mmol) of2,2¹-(ethylenedioxy)-diethylamine (Fluka) and 111 mg (1.31 mmol) ofNaHCO₃ in 7.5 mL of H₂O. The mixture was stirred at room temperature for18 hours. The mixture was partitioned between 50 mL of 1 N HCl and 50 mLof CH₂Cl₂. The CH₂Cl₂ layer was dried (Na₂SO₄), filtered, andconcentrated to provide 1.28 g of viscous oil. Purification by silicagel chromatography (84/15/1 CH₂Cl₂/MeOH/HOAc) gave 670 mg of compound 16as a waxy solid: ¹H NMR (CDCl₃) d 1.32 (m, 16H), 1.49 (m, 16H), 2.46 (m,4H), 2.58 (m, 4H), 3.10-3.23 (m, 16H), 3.34 (m, 4H) 3.48 (m, 4H), 3.53(s, 4H), 3.85 (s, 4H), 4.02 (s, 4H), 5.05 (s, 8H), 5.07 (underlying bdt, 2H), 5.15 (bd t, 2H), 7.30 (m, 20H), 7.40 (bd t, 2H), 8.60 (bd t,2H).

Compound 35: A solution of 613 mg (0.41 mmol) of compound 16 in 20.3 mLof EtOH and 10.1 mL of cyclohexene was stirred and purged with nitrogen.20 mg of 10% Pd on carbon (Aldrich) was added and the mixture was heatedin a 85° oil bath for 1.5 hours. When cool, the mixture was filteredthrough diatomaceous earth using 50/50H₂O/acetone to rinse the flask andfilter. The filtrate was concentrated under vacuum to give 448 mg (114%)of compound 35 as a waxy solid: ¹H NMR (D₂O) d 1.39 (m, 16H), 1.59 (m,16H), 2.57 (t, 4H), 2.65 (t, 4H), 2.88 (t, 8H), 3.23 (t, 4H), 3.29 (t,4H), 3.42 (t, 4H), 3.65 (t, 4H), 3.71 (s, 4H), 4.06 (s, 4H), 4.30 (s,4H).

Compound 17: 546 mg (6.50 mmol) of NaHCO₃ was added to a solution of 445mg (0.406 mmol) of compound 3 in 9.5 mL of H₂O. A solution of 838 mg(3.25 mmol) of compound 10 in 14.4 mL of dioxane was added to theresulting mixture. The mixture was stirred for 7 hours at roomtemperature and partitioned between 50 mL of 0.1 N H₂SO₄ and 50 mL ofCH₂Cl₂. The CH₂Cl₂ layer was discarded, and the aqueous layer wasextracted with two 50 mL portions of CH₂Cl₂, two 50 mL portions of 9/1CH₂Cl₂/MeOH, 50 mL of 4/1 CH₂Cl₂/MeOH, and 50 mL of 3/2 CH₂Cl₂/MeOH. Theextracts were combined and dried (Na₂SO₄), filtered, and concentrated toprovide 282 mg of solid. Crystallization from EtOH/EtOAc/Et₂O gave 143mg (24%) of compound 17 as a white solid: ¹H NMR (CDCl₃/MeOH), d 1.33(m, 16H), 1.55 (s, 16H), 2.55 (m, 8H), 3.21 (m, 16H), 3.39 (s, 4H), 3.55(m, 0.4H), 3.81 (s, 8H), 3.95 (s, 4H), 4.12 (s, 4H). Anal. calc'd forC₅₄H₉₄N₁₂O₁₄Br₄: C, 44.57; H, 6.51; N, 11.55; Br, 21.97. Found: C,45.85; H, 6.49; N, 11.37; Br, 19.90.

Compound 18-[1,5-Bis(N-carbobenzyloxy-6-aminohexanoamido)-3-azapentane]:3.09 g (19.0 mol) of carbonyldiimidazole was added to a solution of 5.05g (19.0 mmol) of N-carbobenzyloxy-6-aminohexanoic acid in 25 mL of EtOAcat room temperature. The mixture was stirred for 15 hours and 1.02 mL(982 mg, 9.52 mmol) of diethylenetriamine was then added followed by2.65 mL (1.93 g, 19.0 mol) of Et₃N. The resulting mixture was stirredfor 4 hours, and the solid product was collected by filtration.Recrystallization (MeOH/EtOAc) gave 4.27 g (75%) of compound 18 as afine grainy solid: m.p. 132-133°; ¹H NMR (CDCl₃) d 1.33 (m, 4H), 1.52(m, 4H), 1.64 (m, 4H), 2.18 (t, 4H), 2.73 (t, 4H), 3.16 (m, 4H), 3.35(m, 4H), 4.96 (bd s, 2H), 5.09 (s, 4H), 6.13 (bd s, 2H), 7.33 (s, 10H);Anal. calc'd for C₃₂H₄₇N₅O₆: C, 64.29; H, 7.50; N, 11.72. Found: C,63.54; H, 7.75; N, 11.91.

Compound 19: 657 uL (880 mg, 3.2 mmol) oftriethyleneglycol-bis-chloroformate (Aldrich) was added to a solution of4.86 g (8.1 mmol) of compound 18 in 162 mL of pyridine in a 20° waterbath. The mixture immediately formed a precipitate. The mixture wasstirred for 16 hours and the resulting cloudy yellow solution wasconcentrated under vacuum. The concentrate was partitioned between 150mL of EtOAc and two 150 mL portions of 1 N HCl (making sure the aqueouslayer was acidic). The aqueous layers were combined and extracted with asecond 150 mL portion of EtOAc. The EtOAc layers were combined, dried(MgSO₄), filtered, and concentrated. The resulting residue wascrystallized (EtOAc/hexanes/CHCl₃) to provide 1.92 g (43%) of compound19 as fine yellow tinted crystals: m.p. 86-91°; ₁H NMR (CDCl₃) 1.31 (m,8H), 1.52 (m, 8H), 1.62 (m, 8H), 2.20 (m, 8H), 3.20 (m, 8H), 3.39 (s,16H), 3.62 (s, 4H), 3.68 (m, 4H), 4.26 (m, 4H), 5.08 (s, 8H), 5.32 (bds, 4H), 7.31 (bd s, 4H), 7.37 (s, 20H); ¹³C NMR (CDCl₃) d 25.1, 26.2,26.4, 29.6, 36.0, 36.2, 38.5, 38.8, 40.8, 64.5, 66.4, 69.1, 70.3, 128.6,128.4, 136.7, 156.5, 156.9, 173.6; Anal. calc'd for C₇₂H₁₀₄N₁₀O₁₈: C,61.87; H, 7.50; N, 10.02. Found: C, 61.68; H, 7.63; N, 9.95.

Compound 36: 3.5 mL of cyclohexene was added to a solution of 800 mg(0.57 mol) of compound 19 in 5 mL of absolute EtOH. The solution wasplaced under nitrogen, 500 mg of 10% Pd on carbon was added, and theresulting mixture was refluxed with stirring for 2 hours. When cool, themixture was filtered through diatomaceous earth and concentrated to give500 mg (100%) of compound 36 as an oil: ¹H NMR (50/50 CDCl₃/CD₃OD) d1.21 (m, 8H), 1.49 (m, 8H), 1.62 (m, 8H), 2.19 (t, J=7.4 Hz, 8H), 2.67(t, J=7.4 Hz, 8H), 3.36 (bd s, 16H), 3.67 (s, 4H), 3.71 (m, 4H), 4.21(m, 4H).

Compound 20: 3.9 g (46.4 mmol) of NaHCO₃ was added to a solution of 5.0g (5.8 mmol) of compound 36 in 37.5 mL of dioxane and 12.5 mL of H₂O.The mixture was cooled to 0° in an ice bath and 8.7 g (34.8 mmol) of4-nitrophenylbromoacetate, compound 10, was added. The mixture wasstirred at 0° for 1 hour and 50 mL of 1 N H₂SO₄ was slowly added. Themixture was extracted with three, 50 mL portions of EtOAc. The EtOAcextracts were discarded and the aqueous layer was extracted with six, 50mL portions of 20/80 MeOH/CH₂Cl₂. The combined MeOH/CH₂Cl₂ layers weredried (Na₂SO₄), filtered, and concentrated. The residue was purified bysilica gel chromatography (step gradient 9/1 CH₂Cl₂/MeOH then 85/15/5CH₂Cl₂/MeOH/THF) to provide 3.62 g (46%) of compound 20 as a whitesolid: melting point 66.0-70.5°. An analytical sample was prepared bypreparative HPLC (C₁₈ reversed phase column, gradient 25/75/0.1 to35/65/0.1 CH₃CN/H₂O/CF₃CO₂H over 50 minutes, 225 nm) to give a clear oilwhich solidified on standing under vacuum to give a white solid: meltingpoint 87-89°; ¹H NMR (CDCl) d 1.35 (m, 8H), 1.55 (m, 8H), 1.64 (m, 8H),2.26 (m, 8H), 3.28 (m, 8H), 3.42 (bd s, 16H), 3.66 (s, 4H), 3.70 (m,4H), 3.89 (s, 8H), 4.19 (m, 4H); ¹³C NMR (CDCl₃) d 25.1, 26.2, 28.8,29.0, 38.5, 39.1, 40.0, 47.8, 48.3, 64.7, 69.1, 70.3, 157.0, 166.3,17.4.9; MS (FAB) m/e (relative intensity) MH+ [1341(25), 1343(60),1345(70), 1347(56), 1349(21)], 705.6(100); Anal. calc'd forC₄₈H₈₄N₁₀O₁₄Br₄: C, 42.86; H, 6.29; N, 9.27; Br, 23.77. Found: C, 42.15;H, 6.28; N, 9.87; Br, 25.33.

Compound 21-[Tetrakis-(2-cyanoethoxymethyl) methane]: This compound wasprepared similarly to the method reported (Bruson, H. A., U.S. Pat. No.2,401,607; Jun. 4, 1946). 27.3 mL (21.8 g, 411 mmol) of acrylonitrilewas added to a stirred solution of 8.0 g (58.8 mmol) of pentaerythritoland 1.76 mL of a 40% aqueous solution of benzyltrimethylammoniumhydroxide in 50 mL of H₂O. A reflux condenser was affixed and themixture was heated under N₂ atmosphere with stirring at 40° for 16 hoursand then at 60° for 24 hours. When cool, the mixture was acidified with1 mL of concentrated HCl and transferred to a separatory funnel. The oilwhich settled to the bottom was collected, and the aqueous phase wasextracted with three 40 mL portions of CH₂Cl₂. The oil and combinedextracts were dried (MgSO₄), filtered, and concentrated to give 23.5 gof oil. Biscyanoethyl ether was removed by Khugelrhor distillation at110° and 0.25 torr. The pot residue was crystallized from 1 L of H₂O togive 8.43 g (41%) of compound 21 as white needles: m.p. 42.5° [Reported(Macromolecules 1991, 24, 1443-1444.) 39-40°]; ¹H NMR (CDCl₃) d 2.61 (t,J=6 Hz, 8H), 3.50 (s, 8H), 3.6 (t, J=6 Hz, 8H).

Compound 22-[Tetrakis-(2-carboxyethoxymethyl) methane]: A solution of5.0 g (14.35 mmol) of compound 21 in 21.5 mL of concentrated HCl wasstirred at 75° for 3 h; during this time a white precipitate formed. Theaqueous HCl was removed under vacuum, and the mixture was concentratedtwice from 25 mL of H₂O. The resulting 9.68 g of solid material wasloaded onto a 45 mm i.d. column containing a 16.5 cm bed of DOW-1-X2resin in the hydroxide form, and the column was eluted with 200 mL ofH₂O followed by 1 N HCl. Fractions containing product, as evidenced byTLC (80/20/1 CH₃CN/H₂O/HOAc), were concentrated to give 1.21 g (21%) of22 as an oil: ¹H NMR (D₂O) d 2.46 (t, J=6 Hz, 8H), 3.22 (s, 8H); 3.55(t, J=6 Hz, 8H).

Compound 23: 3.71 mL (6.06 g, 50.8 mmol) of thionyl chloride was addedto a solution of 1.12 g (2.85 mmol) of compound 22 in 7.0 mL of THF. Themixture was stirred at room temperature for 3 hours and the solventswere removed under vacuum. The crude acid chloride was dissolved in 7 mLof THF. 2.12 mL (1.54 g, 15.24 mmol) of Et₃N was then added to thesolution. The mixture was stirred under N₂ and cooled to 0°. A solutionof 3.60 g (12.74 mmol) of compound 4 in 5 mL of THF was added over a 1minute period. The cooling bath was removed, and the mixture was stirredfor 5.5 hours at room temperature and then partitioned between 25 mL of1 N HCl and four 25 mL portions of EtOAc. The EtOAc layers werecombined, washed with brine, dried (MgSO₄), filtered, and concentratedto provide 3.46 g of viscous oil. Purification by chromatographyon-silica gel (95/5 CH₂Cl₂/MeOH) provided 1.26 g (30%) of compound 23 asa viscous oil: ¹H NMR (CDCl₃) d 2.40 (t, 8H), 3.29 (s, 8H), 3.35 (m,16H), 3.48-3.77 (m, 48H), 5.12 (s, 8H), 5.60 (bd, 4H), 6.85 (bd, 4H),7.34 (s, 20H).

Compound 37: 4.0 mL of cyclohexene and 83 mg of 10% Pd on carbon wereadded to a solution of 142 mg (0.093 mmol) of compound 23 in 8.4 mL ofEtOH under N₂. The mixture was refluxed with stirring in a 90° oil bathfor 3 hours and, when cool, filtered through diatomaceous earth usingCH₂Cl₂ to wash the filter and flask. The filtrate was concentrated toprovide 70 mg (78%) of compound 37 as an oil: 1H NMR (CDCl₃) d 2.90 (t,8H), 3.33 (s, 8H), 3.45 (t, 8H), 3.52-3.73 (m, 48H).

Compound 24: 40 mg (0.48 mmol) of NaHCO₃ and 104 mg (0.40 mmol) ofcompound 10 were added to a solution of 70 mg (0.098 mmol) of compound37 in 2 mL of dioxane and 0.67 mL of H₂O. The mixture was stirred for 17hours at room temperature and 0.5 mL of 1 N H₂SO₄ was added, bringingthe pH to 4. The mixture was concentrated, and the concentrate waspurified by chromatography on G-10 Sephadex® (MeOH). The fractionscontaining product were concentrated under vacuum to provide 91 mg ofoil. Purification of 36 mg of the crude product by HPLC (C₁₈, gradient20/80/0.1 to 35/65/0.1 CH₃CN/H₂O/CF₃CO₂H) gave; 19 mg (44%) of compound24 as an oil: ¹H NMR (CDCl₃) d 2.50 (t, 8H), 3.31 (s, 8H), 3.36-3.72 (m,56H), 3.91 (s, 8H); ¹³C NMR (CDCl₃) d 28.8, 36.5, 39.7, 40.0, 67.2,69.3, 69.5, 70.3, 166.6, 173.0. MS(FAB) m/e (relative intensity) MH+[1425(15), 1427(63), 1429(75), 1431(64), 1433(12)], 577(100).

Compound 25a-[Bis-tolsylate of PEG₃₃₅₀]: 6.47 mL of pyridine was addedto a solution of 16.75 g (5.0 mmol) of polyethylene glycol (J. T. Baker,average molecular weight 3350 g per mol) which had been dried byazeotropic distillation (toluene) in 40 mL of CH₂Cl₂. The solution wasplaced under nitrogen and cooled to 0°. A solution of 7.63 g (40 mmol)of: tosyl chloride in 40 mL of CH₂Cl₂ was added over a 25 minute period.The cooling bath was removed and the mixture was stirred at roomtemperature for 16 hours. The mixture was shaken with 80 mL of 1 N HCland the CH₂Cl₂ layer which contained emulsions was washed with 100 mL ofH₂O. The CH₂Cl₂ layer was dried (MgSO₄), filtered, and concentrated. Theresidue was crystallized from CH₂Cl₂/Et₂O to provide 16.82 g (92%) ofcompound 25a as a white solid: ¹H NMR (CDCl₃) d 2.50 (s, 6H), 3.48 (t,J=5 Hz, 4H), 3.55-3.77 (m, more than 600H, integral too large to beaccurate), 3.83 (t, J=5 Hz, 4H), 7.44 (d, J=7 Hz, 4H), 7.94 (d, J=7 Hz,4H).

Compound 26a-Diazido-PEG₃₃₅₀: A solution of 10.83 g (2.96 mmol) ofcompound 25a and 1.92 g (29.6 mmol) of NaN₃ in 30 mL of DMF was heatedunder N₂ in a 120° oil bath for 3 hours. When cool, the mixture waspartitioned between 100 mL of H₂O and 100 mL of CH₂Cl₂. The CH₂Cl₂ layerwas diluted to 200 mL with CH₂Cl₂ and washed with 100 mL of 1 N HCl,dried (Na₂SO₄), filtered and concentrated. The resulting waxy solid wasrecrystallized. (CH₂Cl₂/Et₂O), and the resulting solids were furtherpurified by chromatography on silica gel (gradient 98/2 to 95/5CH₂Cl₂/MeOH) to provide 4.75 g (47%) of compound 26a as a waxy solid:TLC Rf=0.41 (9/1 CH₂Cl₂); ¹H NMR (CDCl₃) d 3.35 (t, J=5 Hz, 4H), 3.44(t, J=5 Hz, 2H), 3.54-3.77 (m, approx. 300H, integral too large to beaccurate), 3.79 (t, J=5 Hz, 2H).

Compound 27a-[Diamino-PEG₃₃₅₀]: 473 mg of 10% Pd on carbon (Aldrich) wasadded to a solution of 4.75 g (1.39 mmol) of compound 26a in 140 mL ofEtOH. The mixture was shaken under 0.60 psi of H₂ for 30 hours. Becausethe reaction was incomplete (TLC, 9/1 CH₂Cl₂/MeOH), another 473 mg of10% Pd on carbon was added and the mixture was shaken under 60 psi of H₂for another 5 hours. The mixture was then filtered through diatomaceousearth, concentrated under vacuum, and the concentrate was crystallized(CH₂Cl₂/Et₂O) to give 4.03 g (86%) of compound 27a as a white solid: ¹HNMR (CDCl₃) d 2.92 (t, 4H), 3.49 (t, 2H), 3.66 (t, 4H), 3.67 (m, approx.300H, integral too large to be accurate), 3.86 (t, 2H).

Compound 28-[N-hydroxysuccinimidyl ester of compound 7]: 596 mg (2.89mmol) of dicyclohexylcarbodiimide was added to a solution of 1.84 g(2.41 mmol) of compound 7 and 278 mg (2.41 mmol) of NHS in 12 mL of THFat 0° under N₂. The cooling bath was removed, and the mixture wasstirred at room temperature for 16 hours. 250 uL of acetic acid wasadded to the mixture. Stirring was continued at room temperature for 1hour. The mixture was then placed in a freezer for 2 hours. The solidswere removed by filtration, and the filtrate was concentrated to give2.27 g (110%) of crude compound 28 as a viscous oil. Compound 28 wasdifficult to purify without decomposition, so it was used directly toacylate diamino-PEG.

Compound 29a: A solution of 900 mg (1.05 mmol) of compound 28 in 4.68 mLof dioxane was added to a solution of 877 mg (0.26 mmol) of compound 27aand 176 mg (2.10 mmol) of NaHCO₃ in 3.12 mL of H₂O at 0°. The mixturewas stirred for 2 hours and then partitioned between 25 mL of 1 N HCland two 25 mL portions of CH₂Cl₂. The combined CH₂Cl₂ layers were dried(Na₂SO₄), filtered, and concentrated to give a viscous oil. Purificationby silica gel chromatography (gradient, 95/5 to 87/13 CH₂Cl₂/MeOH)yielded 695 mg (55%) of compound 29a as a waxy solid: ¹H NMR (CDCl₃) d2.55 (bd, 8H), 3.39 (m, 16H), 3.44-3.72 (m, approx. 432H, integral toolarge to be accurate), 3.89 (s, 4H), 4.03 (s, 4H), 5.09 (s, 8H), 7.36(s, 20H).

Compound 38a: 7.1 mL of cyclohexene was added to a solution of 688 mg(0.142 mmol) of compound 29a in 14.2 mL of EtOH under N₂. 284 mg of 10%Pd on carbon was added and the resulting mixture was refluxed for 2hours. When cool, the mixture was filtered through diatomaceous earthwith EtOH, and the filtrate was concentrated under vacuum to yield 550mg (90%) of compound 38a as a waxy solid: ¹H NMR (CDCl₃) d 2.58 (m, 8H),2.93 (m, 8H), 3.38-376 (m, approx. 550H), 4.00 (s, 4H), 4.13, (s, 4H).

Compound 30a: A solution of 268 mg (1.04 mmol) of compound 10 in 4.65 mLof dioxane was added to a solution of 550 mg (0.13 mmol) of compound 38aand 175 mg (2.08 mmol) of NaHCO in 3.11 mL of H₂O at 0°. The mixture wasstirred for 20 hours and partitioned between 50 mL of 1 N H₂SO₄ and two50 mL portions of CH₂Cl₂. The combined CH₂Cl₂ layers were dried(Na₂SO₄), filtered, and concentrated to an oil. Purification by G-10Sephadex® chromatography (MeOH) gave an amorphous solid which wascrystallized (EtOH/Et₂O) to provide 378 mg (61%) of compound 30a as awhite solid: ¹H NMR (CDCl₃) d 2.59 (bd s, 8H), 3.38-3.82 (m, approx.500H, integral too large to be accurate), 3.88 (s, 8H), 3.98 (s, 4H),4.10 (s, 4H); bromoacetyl determination (European Journal ofBiochemistry, 1984, 140, 63-71): Calculated, 0.84 mmol/g; Found, 0.50mmol/g.

Compound 25b-[Bis-tosylate of PEG₈₀₀₀]: 2.3 mL (16.5 mmol) oftriethylamine, followed by 3.15 g (16.5 mmol) of TsCl, was added to asolution of 12.0 g (1.5 mmol) of PEG₈₀₀₀ (Aldrich, average molecularweight 8000 g/mmol) which had been dried by azeotropic distillation(toluene) in 30 mL of CH₂Cl₂. The mixture was stirred at roomtemperature for 18 hours and extracted with four, 50 mL portions of 1 NHCl followed by 50 mL of saturated NaCl solution. The CH₂Cl₂ layer wasdried (Na₂SO₄), filtered, and concentrated under vacuum to provide awaxy solid. Recrystallization (CH₂Cl₂/Et₂O) gave 11.0 g (92%) ofcompound 25b as a white solid: ¹H NMR (CDCl₃) d 2.38 (s, 6H), 3.40-3.89(m. approx. 800H, integral too large to be accurate), 4.14 (m, 4H), 7.34(d, J=8.2 Hz, 4H), 7.79 (d, J=8.2 Hz, 4H).

Compound 26b-[Diazido-PEG₈₀₀₀]: 1.86 g (28.6 mmol) of NaN₃ was added toa solution of 10.8 g (1.3 mmol) of compound 25b in 30 mL of dry DMF. Themixture was heated under N₂ at 120° for 2.5 hours. When cool, themixture was partitioned between 240 mL of CH₂Cl₂ and three 50 mLportions of 0.5 N HCl. The CH₂Cl₂ layer was washed with 50 ML ofsaturated NaCl solution, dried (Na₂SO₄) filtered, and concentrated togive a solid. Purification by chromatography on-silica gel (gradient2/98 to 6/94 MeOH/CH₂Cl₂) and recrystallization of th purified product(MeOH/Et₂O) gave 6.95 (66%) of compound 26a as a white solid: TLC(Rf=0.33, 12/88 MeOH/CH₂Cl₂); ¹H NMR (CDCl₃) 3.39-3.86 (m).

Compound 27b-[Diamino-PEG₈₀₀₀]: A solution of 6.9 g (0.86 mmol) ofcompound 26b in 150 mL of MeOH saturated with ammonia was sparged withnitrogen. 1.5 g of 10% Pd/C was added and the mixture was shaken under65 psi of H₂. After 20 hours, TLC analysis indicated that the reactionwas incomplete. As a result, 200 mg of 10% Pd/C was added and shakingunder 65 psi of H₂ was continued for another 20 hours. The mixture wasfiltered through diatomaceous earth and the filtrate was concentratedunder vacuum. The resulting waxy solid was recrystallized (MeOH/Et₂O) togive 6.0 g (89%) of compound 27b as a white solid. ¹H NMR (CDCl₃) d 2.96(t, J=5.1 Hz, 4H), 3.40-3.89 (m, approx. 700H, integral too large to beaccurate).

Compound 29b: 221 mg (2.63 mmol) of NaHCO₃ was added to a solution of3.0 g (0.375 mmol) of compound 27b in 10 mL of water and 3 mL ofdioxane. 1.3 g (1.51 mmol) of compound 28 dissolved in 10 mL of dioxanewas then added. The mixture was stirred for 24 hours and then 40 mL of0.5 N HCl was added. The mixture was extracted with four, 25 mL portionsof CH₂Cl₂. The combined CH₂Cl₂ layers were dried (MgSO₄), filtered, andconcentrated to an oil. Crystallization from MeOH/Et₂O provided 2.0 g(58%) of compound 29b: ¹H NMR (CDCl₃) d 2.52 (m, 8H), 3.40-3.64 (m,approx. 700H, integral too large to be accurate), 3.89 (s, 4H), 4.02 (s,4H), 5.09 (s, 8H), 7.35 (s, 20H).

Compound 38b: 123 mg of 10% Pd/C was added to a solution of 600 mg(0.063 mmol) of compound 29b in 5 mL of absolute EtOH and 2.5 mL ofcyclohexene under nitrogen. This mixture was refluxed under nitrogen for2 hours. The reaction mixture was filtered through diatomaceous earthand evaporated to give 549 mg (97%) of compound 38b as a white solid: ¹HNMR (CDCl₃) d 2.58 (m, 8H), 2.90 (m, 8H), 3.39-3.70 (m, approx. 700H,integral too large to be accurate), 4.05 (s, 4H), 4.15 (s, 4H).

Compound 30b: 100 mg (1.2 mmol) of NaHCO₃, followed by 84 mg (0.32 mmol)of compound 10, was added to a solution of 529 mg (0.059 mmol) ofcompound 38b in 2 mL of dioxane and 5 mL of water. After stirring for 12hours, the reaction was acidified with 1 N H₂SO₄ and extracted withfour, 40 mL of CHCl₃. The combined CHCl₃ layers were dried (MgSO₄),filtered, and concentrated to give 503 mg of semi-solid residue. Theresidue was purified by chromatography on G-10 Sephadex® (MeOH) andcrystallized (MeOH/Et₂O/hexanes) to give 215 mg (39%) of compound 30b asa white solid: ¹H NMR (CDCl₃) d 2.58 (m, 5H, 3.35-3.70 (m, approx. 700H,integral too large to be accurate), 3.89 (s, 8H), 4.01 (s, 4H), 4.16 (s,4H); bromoacetyl determination (European Journal of Biochemistry 1984,140, 63-71): Calculated, 0.42 mmol/g; Found, 0.27 mmol/g.

Compound 31-[PEG₃₃₅₀-bis-chloroformate]: Two drops of dry pyridine,followed by 125 mg (0.418 mmol) of triphosgene, was added to a solutionof 1.0 gram (0.249 mmol) of polyethylene glycol (J. T. Baker, averagemolecular weight 3350 g per mol) which had been dried by azeotropicdistillation (toluene) in 12 mL of CH₂Cl₂. The mixture was stirred atroom temperature for 20 hours and the solvent was evaporated undervacuum to give 1.0 g (100%) of compound 31 as a white solid: ¹H NMR(CDCl₃) d 3.40-3.65 (m, approx. 300H, integral too large to beaccurate), 3.77 (m, 4H), 4.46 (m, 4H).

Compound 32: A solution of 1.0 g (0.25 mmol) of compound 31 in 12 mL of5:1 CH₂Cl₂/dioxane was added dropwise to a 50 solution of 600 mg (1.0mmol) of compound 18 in 10 mL of dioxane and 1.5 mL of pyridine. Theresulting cloudy solution was stirred for 72 hours. 25 mL of CH₂Cl₂ wasadded and the mixture was then filtered. The filtrate was evaporated andthe semi-solid residue was purified by chromatography on G-10 Sephadex®.The resulting solid was crystallized (CH₂Cl₂/Et₂O) to give 829 mg (75%)of compound 32 as a faintly yellow solid: ¹H NMR (CDCl₃) d 1.30 (m, 8H),1.40, (m, 8H), 1.61 (m, 8H), 2.18 (m, 8H), 3.17 (m, 8H), 3.40 (m, 16H),3.62 (m, approx. 300H, integral too large to be accurate), 4.15 (m, 4H),5.07 (s, 8H), 7.33 (m, 20H).

Compound 39: 100 mg of 10% Pd/C was added to a solution of 300 mg (0.065mmol) of compound 32 in 5 mL of absolute EtOH and 2 mL of cyclohexeneunder nitrogen. This mixture was refluxed under nitrogen for 2 hours.The mixture was filtered through diatomaceous earth and the solvent wasevaporated to give 237 mg (90%) of compound 39 as a white solid: ¹H NMR(CDCl₃) d 1.37 (m, 8H), 1.48 (m, 8H), 1.65 (m, 8H), 2.21 (m, 8H), 2.50(m, 8H), 3.39 (m, 16H), 3.64 (m, approx. 300H, integral too large to beaccurate), 4.19 (m, 4H).

Compound 33: 125 mg (0.67 mmol) of NaHCO₃ and 115 mg (0.44 mmol) ofcompound 10 was added to a solution of 225 mg (0.055 mmol) of 39 in 5 mLof dioxane and 5 mL of water. The resulting yellow solution was stirredat room temperature for 12 hours. The solution was then extracted withthree 30 mL portions of CH₂Cl₂. The aqueous layer was acidified with 1 NH₂SO₄ and extracted with three, 30 mL portions of CH₂Cl₂. The combinedCH₂Cl₂ layers were dried (MgSO₄), filtered, and concentrated to providea yellow oil. Purification by chromatography on G-10 Sephadex® (MeOH)and recrystallization of the resulting oil (EtOH/Et₂O) provided 182 mg(7.3%) of compound 33 as a white solid: ¹H NMR (CDCl₃) d 1.35 (m, 8H),1.55 (m, 8H), 1.65 (m, 8H), 2.22 (m, 8H), 3.28 (m, 5H), 3.42 (m, 16H),3.50-364 (m, approx. 300H, integral too large to be accurate), 3.87 (s,8H), 4.18 (m, 4H); bromoacetyl determination (European Journal ofBiochemistry 1984, 140, 63-71): Calculated, 0.87 mmol/g; Found, 0.73mmol/g. Anal Calcd. for C₁₉₁H₃₇₅O₈₇N₁₀Br₄: C, 50.84; H, 8.33; N, 3.09;Br, 7.05. Found: C, 51.98; H, 8.34; N, 2.45; Br, 10.19.

Compound 40-[4-Nitrophenyliodoacetate]: 5.15 g (25 mmol) ofdicyclohexylcarbodiimide and 2.92 g (2.92 mmol) of 4-nitrophenol in 100mL of EtoAc were added to a 0° solution of 3.72 g (20 mmol) ofiodoacetic acid. The mixture was stirred at 0° for 1 hour and at roomtemperature for 2 hours. The solids were removed by filtration, and thefiltrate was concentrated under vacuum. The resulting yellow solid wasrecrystallized (EtOAc/hexanes/trace HOAc) to yield 4.82 g (78%) ofcompound 40 as a yellow-brown solid: ¹H NMR (CDCl₃) d 4.00 (s, 2H), 7.39(d, 2H), 8.40 (m, 2H).

Compound 41: 103 mg (1.22 mmol) of NaHCO₃, followed by 211 mg (0.692mmol) of compound 40, was added to a solution of 110 mg (0.104 mmol) ofcompound 34 in 5 mL of dioxane and 5 mL of H₂O. The mixture was stirredfor 18 hours and then concentrated under vacuum. Purification bychromatography on Sephadex® (MeOH) provided 140 mg (87%) of compound 41as an oil. An analytical sample was prepared by preparative HPLC (C₁₈,gradient 20/80/0.1 to 25/75/0.1 CH₃CN/H₂O/TFA over 60 minutes, 225 nm):¹H NMR (CDCl₃) d 2.59 (m, 4H), 2.65 (m, 4H), 3.44-3.62 (m, 60H), 3.77(s, 4H), 3.78 (s, 4H), 4.02 (s, 4H), 4.21 (s, 4H).

Compound 42: 145 mg (0.935 mmol) of N-methoxycarbonylmaleimide was addedwith vigorous stirring to a solution of 171 mg (0.161 mmol) of compound34 in 8 mL of dioxane, 2 mL of saturated NaHCO₃ solution, and 2 mL ofH₂O at 0° (The Practice of Peptide Synthesis, M. Bodansky and A.Bodansky, Springer-Verlag, New York, 1984, pages 29-31. Keller, O.,Rudinger, J. Helv. Chim. Acta 1975, 5, 531.). After 15 minutes, 25 mL ofdioxane was added, the cooling bath was removed, and stirring wascontinued for 45 minutes at room temperature. The mixture was extractedwith two, 30 mL portions of CHCl₃ and the combined CHCl₃ layers weredried (MgSO₄), filtered, and concentrated to an oil. Purification bychromatography on G-10 Sephadex® (MeOH) gave 103 mg (45%) of compound 42as an oil. An analytical sample was prepared by preparative HPLC (C₁₈,gradient 20/80/0.1 to 25/75/0.1 CH₃CN/H₂O/TFA over 65 minutes, 225 nm)to give an oil: ¹H NMR (CDCl₃) d 2.57 (m, 4H), 2.67 (m, 4H), 3.42-3.65(m, 52H), 3.72 (m, 8H), 4.03 (s, 4H), 4.17 (s, 4H), 6.74 (s, 4H), 6.75(s, 4H).

Compound 43-[hydroxymethyl-tris-(2-cyanoethoxnmethyl)methane]: 0.30 g(5.41 mmol) of KOH, followed by 23 mL (18.6 g, 350 mmol) ofacrylonitrile, was added to a solution of 6.8 g (50 mmol) ofpentaerythritol in 50 mL of H₂O. The mixture was stirred at roomtemperature for 16 hours, acidified with 1.5 mL of concentrated HClsolution, and extracted with two, 50 mL portions of CH₂Cl₂. The combinedCH₂Cl₂ layers were dried (MgSO₄), filtered, and concentrated to give16.97 g of liquid. Purification by chromatography on silica gel (EtOAc)yielded 8.49 g (51%) of compound 43 as a viscous oil: TLC, Rf=0.15(EtOAc); ¹H NMR (CDCl₃) d 2.62 (t, 6H), 3.54 (s, 6H), 3.68 (t, 6H), 3.70(s, 2H).

Compound 44-[hydroxymethyl-tris-(2-carboxymethylethoxymethyl)methane: 78mL of a saturated solution of HCl in MeOH was added to 5.45 g (15.6mmol) of compound 43. The mixture was heated at reflux for 1 hour and,when cool, partitioned between 100 mL of Et₂O and four, 100 mL portionsof Et₂O. The combined Et₂O layers were washed successively with 100 mLof saturated NaHCO₃ solution and 100 mL of saturated NaCl solution,dried (MgSO₄), filtered, and concentrated to yield 4.74 g of viscousliquid. Purification by chromatography on silica gel provided 3.05 g(50%) of compound 44 as an oil: TLC, Rf=0.27 (80/20 EtOAc/hexanes); ¹HNHR (CDCl₃) d 2.58 (t, 6H), 3.43 (s, 6H), 3.61 (s, 2H), 3.69 (t, 6H),3.70 (s, 9H); ¹³C NMR (CDCl₃) d 34.8, 44.9, 51.6, 65.2, 66.9, 71.0,172.1.

Compound 45: A mixture of 560 mg (1.4 mmol) of compound 44 and 1.69 g(6.0 mmol) of compound 4 was heated under nitrogen at 150° for 4 hours.The mixture was partitioned between 50 mL of EtOAc and 25 mL of 1N HCl,and the HCl layer was extracted with 25 mL of saturated NaHCO₃ solution,dried (K₂CO₃), filtered, and concentrated to a viscous residue.Purification by chromatography on silica gel (gradient 95/5 to 90/10CH₂Cl₂/MeOH) provided 300 mg (19%) of compound 45 as a viscous oil: TLC,Rf=0.24 (90/10 CH₂Cl₂); ¹H NMR (CDCl₃) d 2.40 (t, 6H), 3.38 (s, 6H),3.39-3.48 (m, 12H), 3.52-3.67 (m, 32H), 5.13 (s, 6H), 5.62 (bd s, 3H)6.80 (bd s, 3H), 7.40 (s, 15H).

Compound 46: 104 mg of 10% Pd/C was added to a solution of 308 mg (0.269mmol) of compound 45 in 10.4 mL of EtOH and 5.2 mL of cyclohexene undernitrogen. A reflux condenser was attached and the mixture was heated inan 85° 1 oil bath for 1.5 hours. When cool, the mixture was filteredthrough diatomaceous earth and the filtrate was concentrated to provide177 mg of residue. The residue, was partially dissolved in 5.98 mL ofdioxane. The resulting mixture was added to 386 mg (1.49 mmol) ofcompound 10 followed by absolution of 2.51 mg (2.99 mmol) of NaHCO₃ in3.99 mL of H₂O. The resulting mixture was stirred under nitrogen for 18hours and partitioned between 25 mL of 1N HCl and three 25 mL portionsof CH₂Cl₂. The aqueous phase was extracted with three 25 mL portions of3/1 CH₂Cl₂/MeOH and three 25 mL portions of 1/1 CH₂Cl₂/MeOH. The firsttwo CH₂Cl₂ extracts were discarded and the remaining extracts werecombined, dried (Na₂SO₄), filtered, and concentrated to give 102 mg of aviscous oil. Purification by HPLC (C₁₈, 23/77/0.1 CH₃CN/H₂O/CF₃CO₂H, 234nm detection) provided 43 mg (14%) of compound 46 as a viscous oil: ¹HNMR (CDCl₃) d 2.48 (t, 6H), 3.40 (s, 6H), 3.44-3.54 (m, 14H), 3.56-3.62(m, 12H), 3.63 (s, 12H), 3.67 (t, 6H), 3.91 (s, 6H), 6.90 (t, 3H), 7.10(t, 3H); MS (FAB) m/e (relative intensity) MH+ [1103(17), 1105(42),1107(41), 1109(18)], MNa+ [1125(38), 1127(100), 1129(99), 1131(39)].

Compound 47-S-(6-hydroxyhexyl)isothiuronium chloride: 11.1 g (146 mmol)of thiourea was added to a solution of 16.6 mL (20.0 g, 146 mmol) of6-chlorohexanol in 49 mL of ethanol and the mixture was refluxed for 24hours. The mixture was cooled to 0° and the product crystallized. Thecrystals were collected by vacuum filtration and dried to give 28.4 g(92%) of compound 47 as a white solid: mp 122-124°; ¹H-NMR (DMSO) 1.40(m, 4H), 1.65 (m, 2H), 3.21 (t, 2H), 3.41 (t, 2H), 9.27 and 9.33(overlapping broad singlets, 4H); Anal. Calc'd for C₇H₁₇ClN₂O₅: C,39.51; H, 8.06; N, 13.17; S, 15.07. Found: C, 39.69, H, 8.00; N, 13.01;S, 15.16.

Compound 48-6-Mercaptohexan-1-ol: 9.25 g of NaOH pellets was added to asolution of 17.8 mg (83.6 mmol) of compound 47 in 120 mL of H₂O and 120mL of EtOH. The mixture was refluxed for 4 hours. The mixture wascarefully concentrated to approximately 75 mL and the concentrate waspurified by vacuum distillation to provide 7.4 g (66%), of compound 48:bp 95-105° @ 5 mm Hg; ¹H NMR (CDCl₃) 1.41 (m, 9H), 2.59 (dt, 2H), 3.69(t with underlying brd s, 3H); ¹³C NMR (CDCl₃) d 24.5, 25.2, 28.0, 32.5,33.9, 62.7; Anal. calc'd for C₆H₁₄OS: C, 53.68, H, 10.51; S, 23.89.Found: C, 53.35; H, 10.72; S, 23.60.

Compound 49-Bis-(6-hydroxyhexyl)disulfide: A solution of 4.02 g (15.8mmol) of I₂ in 90 mL of MeOH was added dropwise over a period of 10minutes to a solution of 4.26 g (31.7 mmol) of compound 48 in 10 mL ofMeOH and 13.7 mL (9.97 g, 98.5 mmol) of Et₃N under N₂ atmosphere andcooled in an ice bath. The cooling bath was removed and the mixture wasstirred at ambient temperature for 4 hours. The mixture was concentratedon the rotary evaporator and purified by silica gel chromatography (1:1hexane/EtOAc) to provide 3.12 g (73%) of compound 4 as a pale yellowsolid: TLC R_(f) 0.18 (1:1 hexane/EtOAc); mp 38-48°; ¹H NMR (CDCl₁)1.15-2.20 (m, 16H), 2.73 (t, 4H), 3.70 (t, 4H); Anal. calc'd forC₁₂H₂₆S₂O₂: C, 54.09; H, 9.84; S, 24.06. Found: C, 54.85, H, 9.86; S,24.11.

Compound50-Mono-O-(4′,4″-dimethoxytriphenylmethyl)-bis-(6-hydroxyhexyl)disulfide:3.97 g (11.7 mmol) of 4,4′-dimethoxytriphenylmethyl chloride was addedto a solution of 3.12 g (11.7 mmol) of compound 49 and 45 mL ofpyridine. The mixture was stirred at ambient temperature for 16 hours.Most of the pyridine was removed on the rotary-evaporator and theresidue was partitioned between 100 mL of saturated NaHCO₃ solution and100 mL of EtOAc. The EtOAc layer was washed with 50 mL of saturated NaClsolution, dried (Na₂SO₄), filtered and concentrated to an oil.Purification by silica-gel chromatography (9:1 CH₂Cl₂/EtOAc) yielded2.84 g (43%) of compound 5 as a viscous oil: TLC R_(f) 0.35 (9:1CH₂Cl₂/EtOAc); ¹H NMR (CDCl₃) 1.41 (m, 8H), 1.65 (m, 8H), 2.70 (twooverlapping triplets, 4H), 3.08 (t, 2H), 3.65 (t, 2H), 3.81 (s, 6H),6.85 (d, 4H), 7.32 (m, 7H), 7.47 (d, 2H); HRMS (FAB, M+), calc'd forC₃₃H₄₄O₄S₂: 568.2681. Found: 568.2665.

Compound51-O-[14-(4′,4″-Dimethyoxytriphenylmethoxy)-7,8-dithiotetradecyl]-O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite:458 mg (1.52 mmol) ofO-cyanoethyl-N,N,N′,N′-tetra-isopropylphosphorodiamidite in 0.5 mL ofCH₂Cl₂ was added to a solution of 771 mg (1.36 mmol) of compound 50 and116 mg (0.68 mmol) of diisopropylammonium tetrazolide in 6.8 mL ofCH₂Cl₂ under N₂ atmosphere. The mixture was stirred for 4 hours andpartitioned between 25 mL of NaHCO₃ and 3×25 mL of CH₂Cl₂. The combinedCH₂Cl₂ layers were washed with saturated NaCl solution, dried (Na₂CO₃),filtered and concentrated to an oil. Purification by filtration througha 2″ plug of basic alumina in a 25 mm column, eluting with 9:1CH₂Cl₂/Et₃N provided 831 mg (80%) of compound 51 as a viscous oil: ¹HNMR (CDCl₃) d 1.25 (m, 12H), 1.45 (m, 8H), 1.70 (m, 8H), 2.72 (m, 6H),3.09 (t, 2H), 3.65 (m, 4H), 3.87 (s, 6H), 3.91 (m, 2H), 6.89 (d, 4H),7.35 (m, 7H), 7.49 (d, 2H); ³¹P NMR (CDCl₃ with 15% H₃PO₄ internalstandard) 147.69; HRMS (FAB, MH+) calc'd for C₄₂H₆₂N₂O₅PS₂ 769.3839,found 769.3853.

Compound 52-Trityl-HAD alcohol: 60 g (0.21 mol) of trityl chloride wasadded to a solution of 57 g (0.21 mole) of compound 49 and 60 mL ofpyridine. This mixture was stirred at 100° C. for 19 hours. The reactionmixture was cooled to room temperature and filtered. The filtrate wasdiluted with 300 mL of methylene chloride and extracted by 200 mL ofsaturated sodium bicarbonate. The organic layer was dried over Na₂SO₄,filtered and concentrated to an oil. Purification by silica gelchromatography (gradient 9:1 hexanes:ethyl acetate 3:1 hexanes:ethylacetate) yielded 55 g of compound 52 (50%): ¹H NMR (CDCl₃) δ 1.38 (m,8H), 1.63 (m, 8H), 2.66 (m, 4H), 3.04 (t, 2H), 3.62 (t, 2H), 7.25 (m,9H), 7.42 (m, 6H). HRMS (FAB, M+) calc'd for C₃₁H₄₀O₂S 508.2470, found508.2482.

Compound 53-Trityl HAD Phosphoramidite: To a solution of 10 g (19.7mmol) of compound 52 and 6.3 mL (36.2 mmol) of diisopropylethylamine in90 mL of methylene chloride at 0° C. under argon was slowly added 4.5 mL(20.2 mmol) of 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. Afterstirring for 90 minutes, the reaction mixture was extracted twice with100 mL of saturated sodium bicarbonate. The methylene chloride solutionwas dried over Na₂SO₄, filtered and concentrated to an oil. Purificationby basic alumina chromatography (75:24:1, hexanes:ethylacetate:triethylamine) provided 11.3 g (81%) of compound 53 as an oil:¹H NMR (CDCl₃) δ 1.18 (m, 12H), 1.37 (m, 8H), 1.62 (m, 8H), 2.6 (m, 6H),3.04 (t, 2H), 3.60 (m, 4H), 3.82 (m, 2H), 7.26 (m, 6H), 7.44 (m, 9H).HRMS (FAB, MH+) calc'd for C₄₀H₅₈N₂O₃PS₂ 709.3626, found 709.3621.

Compound 54-O(tert-butyldimethylsilyl)-5-hexenol: 15.66 g (230 mmol) ofimidazole and 20.0 g (130 mmol) of tert-butyldimethylsilyl chloride wereadded to a solution of 12.47 mL (10.4 g, 104 mmol) of 5-hexene-1-ol in104 mL of DMF. The mixture was stirred at ambient temperature for 4hours and partitioned between 200 mL of EtOAc and 100 mL of saturatedNaHCO₃ solution. The EtOAc layer was washed with 100 mL of saturatedNaHCO₃ solution, 100 mL of saturated NaCl solution, dried (MgSO₄),filtered, and concentrated to a volume of approximately 100 mL.Distillation under vacuum provided 70.07 g. (90%) of compound 54: bp130-143° @ 100 mm Hg; ¹H NMR (CDCl₃) 0.11 (s, 6H), 0.95 (s, 9H), 1.48(m, 2H), 1.57 (m, 2H), 2.11 (dt, 2H), 3.66 (t, 2H), 5.03 (m, 2H), 5.86(m, 1H); ¹³C NMR (CDCl₃) −5.25, 18.40, 25.21, 26.01, 32.35, 33.60,63.09, 114.40, 138.92; Anal. calc'd for C₁₂H₂₆OSi: C, 67.22; H, 12.22.Found: C, 66.96; H, 12.16.

Compound 55-1-O-(tert-butyldimethylsilyl)-1,5,6-hexanetriol: To asolution of 9.86 g (46.0 mmol) of compound 54 in 92 mL of acetone wasadded a solution of 6.46 g-(55.2 mmol) of N-methylmorphoholine oxide in23 mL of H₂O. To the mixture was added 443 μl of a 2.5% solution of OsO₄in tert-butyl alcohol (360 mg of solution, 9.0 mg Of OsO₄, 35 μmol) and50 μL of 30% H₂O₂. The mixture was stirred for 16 h and a solution of474 mg of sodium dithionite in 14 mL of H₂O was added. After another 0.5h the mixture was filtered through celite. The filtrate was dried withMgSO₄ and filtered through 1″ of silica gel in a 150 mL Buchner funnelusing 250 mL portions of EtOAc to elute. Fractions containing productwere concentrated to provide 11.0 g (96%) of as a viscous oil: TLC R_(f)0.2 (1:1 hexane/EtOAc); ¹H NMR (CDCl₃) 0.05 (s, 6H), 0.89 (s, 9H), 1.25(m, 4H), 1.55 (m, 2H), 3.41 (dd, 2H), 3.62 (t, 2H), 3.71 (m, 1H); ¹³CNMR (CDCl₃) −5.23, 18.42, 21.91, 26.02, 32.68, 32.81, 63.16, 66.74,72.24; HRMS (FAB, NH+), calc'd for C₁₂H₂₉O₃Si: 249.1886. Found:249.1889.

Compound56-5,6-(bis-O-benzoyl)-1-O-(tert-butyldimethylsilyl)-1,5,6-hexanetriol:6.18 mL (7.48 g, 53.2 mmol) of benzoyl chloride was added to a solutionof 5.29 g (21.3 mmol) of 55 in 106 mL of pyridine. The mixture wasstirred for 18 hours and concentrated on the rotary evaporator. Themixture was partitioned between 100 mL of cold 1 N HCl and 100 mL ofEtOAc. The pH of the aqueous layer was checked to make sure it wasacidic. The EtOAc layer was washed successively with 100 mL of H₂O and100 mL of saturated NaCl, dried (MgSO₄), filtered, and concentrated toprovide 10.33 g (99%) of compound 56 as a viscous yellow oil: TLC R_(f)0.45 (1:4 EtOAc/hexanes); ¹H NMR (CDCl₃) d 0.05 (s, 6H), 0.88 (s, 9H),1.59 (m, 4H), 1.85 (m, 2H), 3.14 (t, 2H), 4.49 (dd, 1H), 4.59 (dd, 1H),5.54 (m, 1H), 7.45 (m, 4H), 7.58 (m, 2H), 8.05 (m, 4H).

Compound 57-5,6-(bis-O-benzoyl-1,5,6-hexanetriol: 10.7 mL (10.7 mmol) of1 N tetrabutylammonium fluoride in THF was added to a solution of 2.62 g(5.36 mmol) of compound 56 in 10.9 mL of THF. The mixture was stirredfor 16 hours. The mixture was partitioned between 25 mL of saturatedNaHCO₃ solution and 3×25 mL of EtOAc. The combined EtOAc extracts werewashed with saturated NaCl solution, dried (MgSO₄), filtered andconcentrated to a viscous oil which was purified by silica gelchromatography (1:1 hexane/EtOAc) to provide 823 mg (41%) of compound 57as a viscous oil; R_(f) 0.14 (1:1 hexane/EtOAc); ¹H NMR (CDCl₃) d 1.58(m, 2H), 1.68 (m, 2H), 1.88 (m, 2H), 3.68 (t, 2H), 4.52 (dd, 1H), 4.62(dd, 1H), 5.56 (m, 1H), 7.46 (m, 4H), 7.58 (m, 2H), 8.05 (m, 4H); ¹³CNMR (CDCl₃) d 22.08, 31.20, 31.30, 32.88, 62.92, 66.17, 72.63, 128.93,130.19, 130.57, 133.62, 166.72, 166.86; HRMS (FAB MH+), calc'd forC₂₀H₂₃O₅; 343.1545. Found: 343.1553.

Compound 58-O-[5,6-(bis-O-benzoyloxy)-hexyl]-O-(2-cyanoethyl)-NN-diisopropylphosphoramidite: A solution of 989 mg (3.28 mmol) ofO-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidit in 2.0 mL ofCH₂Cl₂ was added to a solution of 1.02 g (2.98 mmol) of compound 57 and255 mg (1.49 mmol) of diisopropylammonium tetrazolide (prepared bymixing acetonitrile solutions of diisopropylamine and tetrazole in aone-to-one mole ratio and concentrating to a white solid) in 14.9 mL ofCH₂Cl₂. The mixture was stirred for 4.1 hours and then partitionedbetween 25 mL of CH₂Cl₂ and 25 mL of chilled saturated NaCO₃ solution.The CH₂Cl₂ layer was washed with saturated NaCl solution, dried(Na₂SO₄), filtered, and concentrated. Purification by filtration througha 2″ plug of basic alumina in a 25 mm column, eluting with 9:1EtOAc/Et₃N, provided 1.5 g (93%) of compound 58 as a viscous oil: ¹H NMR(CDCl₃) d 1.19 (m, 12H), 1.62 (m, 2H), 1.73 (m, 2H), 1.90 (m, 2H), 2.62(dd, 2H), 3.53-3.92 (m, 6H), 4.53 (dd, 1H), 4.62 (dd, 1H), 5.58 (m, 1H),7.48 (m, 4H), 7.60 (m, 2H), 8.09 (m, 4H); ³¹P NMR (CDCl₃ with 15% H₃PO₄internal standard) d 148.2; HRMS (FAB, MH+), calc'd for C₂₉H₄₀O₆N₂P543.2624. Found, 543.2619.

Compound 59-[4(iodoacetamido)benzoic acid: This compound was prepared asdescribed by Weltman, J. K., 1983 Biotechniques 1:148-152. Briefly, 708mg (2.0 mmol) of iodoacetic anhydride was added to a solution of 137 mg(1.0 mmol) of para-aminobenzoic acid in 10 mL of dioxane. The mixturewas stirred in the dark for 18 hours and partitioned between 25 mL ofH₂O and 25 mL of EtOAc. The EtOAc layer was washed with saturated NaClsolution, dried (MgSO₄), filtered and, concentrated to yield 797 mg of apeach colored solid. Recrystallization from hexanes/EtOAc yielded 221 mg(72%) of 4-(iodoacetamido)benzoic acid as a white solid: mp '220-230°;¹H NMR (CDCl₃) d 3.86 (s, 2H), 7.68 (d, 2H), 7.91 (d, 2H), 10.60 (s,1H).

Compound 60-[4-(iodoacetamido)benzoyl derivative ofα,ω-bis-(N-2-aminoethylcarbamoyl)polyethyleneglycol: 188 mg (0.909 mmol)of dicyclohexylcarbodiimide was added to a solution of 185 mg (0.606mmol) of 4-(iodoacetamido)benzoic acid and 406 mg (0.121 mmol) ofα,ω-bis-(N-2-aminoethylcarbamoyl)polyethyleneglycol (Sigma Chemical Co.,St. Louis, Mo., dried by azeotropic distillation with toluene) in 2 mLof THF. The mixture was stirred for 2 hours and then six drops of aceticacid were added. 10 mL of CH₂Cl₂ was added and the mixture was kept in afreezer for 30 minutes. The mixture was filtered to remove solids andthe filtrate was concentrated to a viscous residue. Purification bysilica gel chromatography (gradient 99/1 to 96/4 CH₂Cl₂/MeOH) provided asolid which was triturated with MeOH to give 292 mg of a cream coloredsolid: ¹H (CDCl₃) 3.48 (m, 8H), 3.63 (bd s, (CH₂CH₂O)_(n), integral toolarge to integrate), 3.98 (s, 4H), 4.18 (bd m, 4H), 5.91 (bd m, 2H),7.48 (bd m, 2H), 7.76 (d, 4H), 7.88 (d, 4H), 9.38 (bd m, 2H): iodoacetyldetermination (European Journal of Biochemistry 1984, 140, 63-71):Calculated, 0.46 mmol/g; Found, 0.37 mmol/g.

EXAMPLE 3 Preparation of Activated Valency Platform Molecules andConjugates

There are many ways to form conjugates of biological or chemicalmolecules and valency platform molecules. A particularly specific methoduses a thiol attached to the biological or chemical molecule to reactnucleophilically with a reactive “thiophillic” group on the valencyplatform molecule to form a thioether bond, but other combinations ofreactive groups on the platform molecule and on the biological orchemical molecule can also be employed for attaching biological orchemical molecules covalently to a valency-platform molecule. Table 1contains a number of combinations of mutually reactive groups. Thepreference of any given method is dictated by the nature of thebiological or chemical molecule (solubility presence of other reactivegroups, etc.). TABLE 1 Nucleophile Mutually Reactive Group amine,hydrazide active ester, anhydride, acid hydrazine halide, sulfonylhalide, imidate ester, isocyanate, isothiocyanate, chloroformatecarbodiimide adduct, aldehyde, ketone sulfhydryl haloacetyl, alkylhalide, alkyl sulfonate, maleimide, α,β- unsaturated carbonyl, alkylmercurial, sulfhydryl, α,β- unsaturated sulfone

The following examples illustrate how-various valency-platform moleculescan be synthesized and conjugated with biological or chemical molecules.These examples show how peptides and oligonucleotides can be conjugatedto valency platform molecules using some of the mutually reactive groupsin Table 1. In addition to peptides and oligonucleotides, otherbiologically active molecules (proteins, drugs, etc.) can also beconjugated to valency platform molecules.Combination 1: Thiol on Platform—Thiophile on Ligand

Compound A: Compound 36 (861 mg, 1.0 mmol) and 252 mg (3.0 mmol) ofNaHCO₃ are dissolved in 20 mL of 1/1 dioxane/H₂O. The mixture is cooledto 0°, and a solution of 1.16 g (5.0 mmol) ofN-succinimidyl-S-acetylthioacetate (Prochem Inc.) in 40 mL of dioxane isadded to the stirred mixture. After 1 hour the mixture is extracted withCH₂Cl₂. The combined extracts are dried (MgSO₄), filtered, andconcentrated. The crude product is purified by silica gel chromatographyto provide A.

Compound B—Platform with Four Thiol Groups. A solution of 732 mg (0.55mmol) of A in 7.3 mL of DMSO is added to 55 mL of helium sparged pH 10,100 mM sodium carbonate, 10 mM NH₂OH buffer. The mixture is kept underN₂ and stirred for 1 hour to obtain an approximately 1.0 m solution oftetra-thiol platform B.

Compound X—Bromoacetylated Peptide: A peptide is synthesized withstandard solid phase methods on a Wang (p-alkoxybenzyl) resin using FMOCchemistry. FMOC protected amino acids are added sequentially to theamino terminus. The final step involves couplingN-bromoacetylaminocaproic acid. The protecting groups are removed, andthe peptide is removed from the resin with trifluoroacetic acid to giveX which is purified by preparative reverse phase HPLC.

Peptide—Platform Conjugate, C. To the approximately 10 mmol solution oftetrathiol platform, B, in pH 10 buffer, is added an excess of asolution of bromoacetylated peptide, X, in DMSO. The peptide conjugate,C, is purified by preparative reverse phase HPLC.Combination 2: Amine on Platform—Activated Carboxylate on Peptide

Compound Y—Peptide with Activated Carboxylate. A peptide is synthesizedwith standard solid phase methods on a Wang (p-alkoxybenzyl) resin,using TFA stable protecting groups (benzyl ester on carboxyl groups andCBZ on amino groups). Amino acid residues are added sequentially to theamino terminus. The peptide is removed from the resin with TFA toprovide a peptide with one free carboxyl group at the carboxy terminusand all the other carboxyls and amines blocked. The protected peptide,Y, is purified by reverse phase HPLC.

Peptide—Platform Conjugate, D. Compound Y (0.3 mmol) is dissolved in 1mL of DMF, and to the solution is added 0.3 mmol ofdiisopropylcarbodiimide and 0.3 mmol of HOBT. The solution is added to asolution of 0.025 mmol tetraamino platform, 36, in 1 mL of DMF. Whencomplete, the DMF is removed under vacuum to yield a crude fullyprotected conjugate. The conjugate is dissolved in MeOH, and thesolution is placed in a Parr hydrogenation apparatus with 100 mg of 10%Pd/C per gram of conjugate. The mixture is shaken under 60 psi H₂, andthe deprotected conjugate, D, is purified by preparative reverse phaseHPLC.Combination 3: Amine on Platform—Aldehyde on Oligonucleotide

Oligonucleotide—Platform Conjugate, E. A 500 uL aliquot (100 umol) of a200 mM solution of NalO₄ is added to a solution of 1.0 g (400 mg of fulllength, 25 mmol) of ACT-modified (CA)₂₅ in 19.5 mL of H₂O at 0° in thedark. The mixture is kept at 0° for 40 minutes, and 50 mL of EtOH isadded. The mixture is kept at −20° for 30 minutes and centrifuged for 5minutes at 2000 RPM. The supernatant is discarded, and the pellet isdried under vacuum. The pellet is dissolved in 3.3 mL of H₂O, and to theresulting solution is added a solution of 4.3 mg (0.005 mmol) of 36 in2.0 mL of pH 8.0 100 mM sodium borate. To the resulting-solution isadded 250 uL (50 mmol) of a 200 mM solution of pyridine-borane complexin MeOH, and the mixture is kept at 370 for 4 days. The conjugate, E,can be purified by ion exchange chromatography.Combination 4: Activated Carboxylate on Platform—Amine on Ligand

Compound F—Platform with Four Carboxylic Acid Groups. Succinic anhydride(1.0 g, 10 mmol) is added to a solution of 861 mg (1.0 mmol) of 36 and252 mg (3.0 mmol) of NaHCO₃ in 20 mL of 1/1 dioxane/H₂O, and the mixtureis stirred for 16 h at room temperature. The mixture is acidified with 1N HCl and concentrated. The concentrate is purified by silica gelchromatography to provide F.

Compound G—Platform with Four N-Hydroxysuccinimidyl Esters. A solutionof 126 mg (0.1 mmol) of F and 46 mg (0.4 mmol) of N-hydroxysuccinimidein 5 mL of anhydrous THF is prepared. The mixture is cooled to 0° and103 mg (0.5 mmol) of dicyclohexylcarbodiimide is added. The mixture isstirred allowing to come to room temperature over several hours. Thesolids are removed by filtration, and the filtrate is concentrated toprovide G which can be purified by silica gel chromatography.

Compound Z—Peptide with Amino Group. A peptide is synthesized withstandard solid phase methods on a Wang (p-alkoxybenzyl) resin. Lysineε-amines are protected as CBZ groups. Amino acid residues areadded-sequentially to the amino terminus using FMOC chemistry. The lastresidue added is N-FMOC-aminocaproic acid. After cleaving from the resinwith trifluoroacetic acid, the FMOC group is removed with piperidine toprovide a peptide with a free amine linker. The peptide, Z, is purifiedby reverse phase HPLC.

Peptide—Platform Conjugate, H. A solution of 0.05 mmol of Z and 0.1 mmolof Et₃N in 1 mL of DMF is prepared. To the solution is added a solutionof 16.5 mg (0.01 mmol) of G in 1 mL of DMF. The mixture is stirred untilthe reaction is complete. To remove protecting groups, the conjugate isdissolved in MeOH, and the solution is placed in a Parr hydrogenationapparatus with 100 mg of 10% Pd/c per gram of conjugate. The mixture isshaken under 60 psi H₂, and the deprotected conjugate, H, is purified bypreparative reverse phase HPLC.Combination 5: Isothiocyanate on Platform—Amine on Ligand

Compound 1—Platform with Four Isothiocyanates. Thiophosgene (381 uL, 575mg, 5.0 mmol) is added to a solution of 861 mg (1.0 mmol) of 36 in 10 mLof THF, and the mixture is stirred at room temperature until, completeby TLC. The mixture is partitioned between methylene chloride and asolution of 5% NaHCO₃. The extracts are dried (MgSO₄), filtered, andconcentrated. The product, I, is purified by silica gel chromatography.

Peptide—Platform Conjugate, J. A solution of 0.05 mmol of Z and 0.1 mmolof Et₃N in 1 mL of DMF is prepared. To the solution is added a solutionof 10.3 mg (0.01 mmol) of I in 1 mL of DMF. The mixture is stirred untilthe reaction is complete. To remove protecting groups, the conjugate isdissolved in MeOH, and the solution is placed in a Parr hydrogenationapparatus with 100 mg of 10% Pd/C per gram of conjugate. The mixture isshaken under 60 psi H₂, and the deprotected conjugate, J, is purified bypreparative reverse phase HPLC.Combination 6: Chloroformate on Platform—Amine on Ligand

Compound K—Platform with Four Hydroxyl Groups. A solution of 205 uL (275mg, 1 mmol) of triethylene glycol bis-chloroformate in 5 mL of CH₂Cl₂ isadded to a solution of 497 uL (525 mg, 5 mmol) of diethanolamine and 696uL (506 mg, 5 mmol) of Et₃N in 5 mL of CH₂Cl₂ at 0°. The mixture isallowed to warm to room temperature and stirred until complete asevidenced by TLC. The mixture is concentrated and the product, K, isisolated by silica gel chromatography.

Compound L—Platform with Chloroformate Groups. Pyridine (100 ul)followed by 1.19 g (4 mmol) of triphosgene are added to a solution of412 mg (1 mmol) of K in 20 mL of CH₂Cl₂. The mixture was stirred at roomtemperature for 20 hours, and the solvent was evaporated, under vacuumto give compound L.

Peptide—Platform Conjugate, M. A solution of 1 mmol of Z in 10 mL ofpyridine is added to a solution of 132 mg (0.2 mmol) of L in 5 mL of 1/1THF/pyridine. The mixture is stirred until the reaction is complete.Solvents are removed in vacuo. To remove protecting groups, theconjugate is dissolved in MeOH, and the solution is placed in a Parrhydrogenation apparatus with 100 mg of 10% Pd/C per gram of conjugate.The mixture is shaken under 60 psi H₂, and the deprotected conjugate, M,is purified by preparative reverse phase HPLC.

EXAMPLE 4 Synthesis of Conjugates Comprising Two Different BiologicalMolecules

It can be useful to conjugate more than one kind of biologically activegroup to a platform molecule. This example describes the preparation ofa platform containing two maleimide groups, which react with athiol-containing peptide, and two activated ester groups, which reactwith a drug containing a free amine. The resulting conjugate containstwo peptides and two drug molecules as shown in Scheme 20.

Preparation of heteroactivated valency platform molecule Benzyl6-amniocaproate tosylate salt, K: A mixture of 32 mmol of 6-aminocaproicacid, 51 mmol of p-toluenesulfonic acid, and 40 mmol of benzyl alcoholin 60 mL of toluene is refluxed using a Dean-Stark trap to remove water.When the reaction is complete, the mixture is cooled, and the productprecipitates. The solid is collected by filtration, and recrystallizedfrom EtOH/Et₂O to provide compound K.

Compound L: Dicyclohexylcarbodiimide (2 equivalents) is added to asolution of 1 equivalent of compound 5 and 2 equivalents ofN-hydroxysuccinimide in THF. The mixture is stirred for 4 hours and 2.2equivalents of compound E is added. The mixture is stirred until thereaction is complete as evidenced by TLC. The mixture is filtered andconcentrated. The product is purified by silica gel chromatography.

Compound M: Compound L is treated with trifluoroacetic acid in CH₂Cl₂.When the reaction is complete, the mixture is concentrated under vacuumto provide compound M as the trifluoroacetate salt.

Compound N: Compound 6 (Scheme 2) is treated with trifluoroacetic acidin CH₂Cl₂. When the reaction is complete, the mixture is concentratedunder vacuum to provide compound N as the trifluoroacetate salt.

Compound O: 3.2 mmol of triethyleneglycol bis-chloroformate is added toa solution of 4 mmol of compound M and 4 mmol of compound N in 162 mL ofpyridine in a 20° water bath. The mixture is stirred until complete byTLC and concentrated under vacuum. The concentrate is dissolved inCH₂Cl₂ and washed successively with 1 N HCl solution, 5% NaHCO₃solution, and saturated NaCl solution. The CH₂Cl₂ layer is dried(MgSO₄), filtered and concentrated. The concentrate is dissolved in 10mL of EtOH and 10 mL of 1 M NaOH is added. The mixture is stirred forseveral hours, until no further reaction appears to take place by TLC.The mixture is acidified to pH 1 with 1 N HCl and extracted with CH₂Cl₂.The CH₂Cl₂ layer is dried. (MgSO₄), filtered, and concentrated. Theproduct, O, is isolated by silica gel chromatography.

Compound P: Compound O is dissolved in EtOH and so hydrogenated in aParr shaker with 100 mg of 10% palladium on carbon per gram of O. Thereaction is monitored for completeness by TLC. When the reaction iscomplete, the catalyst is removed by filtration, and the mixture isconcentrated to yield compound P.

Compound Q: 3 mmol of N-methoxycarbonylmaleimide is added to a solutionof 1 mmol of compound k in 20 mL of dioxane and 5 mL of saturated NaHCO₃at 0°. The mixture is stirred for an hour, acidified with 1 N HCl, andextracted with CH₂Cl₂. The CH₂Cl₂ layer is dried (MgSO₄), filtered, andconcentrated, and the product is purified by silica gel chromatographyto yield Q.

Compound R: 2 mmol of DCC is added to a solution of 1 mmol of Q and 2mmol of p-nitrophenol in CH₂Cl₂ and the mixture is stirred for 16 h. Thesolids are removed by filtration, and the filtrate is concentrated andpurified by silica gel chromatography to yield B.

Conjugate with two peptides and two drug molecules, compound S: Anexcess of two equivalent of thiol-containing peptide is added to asolution of 1 equivalent of heteroactivated platform, R, in pH 7.5phosphate buffer. The mixture is stirred for 1 hour, and excess of twoequivalents of amine-containing drug is added. The conjugate, S, isisolated by reverse-phase HPLC or ion-exchange chromatography or acombination of both.

EXAMPLE 5 Synthesis and Testing of Conjugate 3-II

Preparation of DMTr-5′-Modified (CA)₁₀.

The polynucleotide d-[DMTr-(bzCp(CE)bzA)₁₀] was prepared on a Milligen8800 Prep Scale DNA synthesizer (See FIG. 6A) following themanufacturer's protocols for DNA phosphoramidite synthesis. Thesynthesis was carried out on 10 g of DMTr-d-bzA-CPG support with anucleoside loading of 30.0 μmol/g. The final DMTr blocking group wasremoved using the machine protocol. Milligen activator solution, Cat.No. MBS 5040 (45 mL) and 0.385 g of compound 51 (see Reaction Scheme 11)were added to the reaction and the suspension was mixed for 8 minutes byargon ebullition. The mixture was oxidized by the usual machine protocoland the support-bound polynucleotide was collected by filtration, airdried and treated with 100 mL of concentrated ammonia for 16 hours at55° C. When cool, the mixture was filtered through a Gelman 10μpolypropylene filter. The filter was washed with 200 mL of 2 mM NaCladjusted to pH 12 with NaOH. The filtrate was then applied to a Amiconchromatography column (0.45×9.4 cm, 150 mL) which had been packed withQ-Sepharose (Pharmacia) equilibrated first with 3M NaCl and then with 2mM NaCl, pH 12. The column was eluted with 500 mL of a linear gradient(2 mM NaCl, pH 12 to mL 1.3 M NaCl, pH 12), then washed with 1.3 M NaCl,pH 12 until all U.V. absorbing material came off. Fractions whichabsorbed at 260 nm were further analyzed by polyacrylamideelectrophoresis and those containing pure product were pooled. The pool(120 mL) was treated with 240 mL of cold isopropanol and stored for 30minutes at −20° C. The precipitate was collected by centrifugation in aSorvall RC 3B centrifuge using a model H-6000A rotor for 15 minutes at3000 rpm and 4° C. to yield DMTr-5′-modified (CA)₁₀ (14946 A₂₆₀ units,498 mg, 62.2 μM, 20% based on 300 μM, CPG nucleoside.)Synthesis of a Tr-5′-Modified (CA)₁₀

The synthesis of Tr-5′-modified (CA)₁₀ was carried out as describedabove for the synthesis of DMTr-5′-modified (CA)₁₀ (prepared asdescribed in Reaction Scheme 11) by substituting compound 53 forcompound 51.

Conjugation of DMTr-5′Modified Polynucleotides to Compound 3(IA-DABA-PEG, Reaction Scheme 1)—Preparation of Conjugate 3-I

In the conjugation procedures that follow, all the buffers and solutionsemployed were thoroughly sparged with helium and all reaction vesselswere purged with argon before use. A solution of 11,568 A₂₆₀ units (48.2μmol, assume molar extinction at 260 nm=240,000) of the DMTr-5′-modified(CA)₁₀ in 7.7 mL water was treated with 1 mL of 0.1 M NaHCO₃ and 210 μL(876 μmol, 18 times molar excess) tributylphosphine for 0.5 hour at roomtemperature. The suspension was shaken from time to time. The suspensionwas treated with 0.8 mL of 3M NaCl and 16 mL of cold isopropanol. After30 minutes at −20° C., the material was centrifuged at 3000 rpm for 20minutes. The pellet was redissolved in 2 mL of water, 0.2 mL of 3M NaCl,treated with 4 mL isopropanol and recentrifuged. The pellet was brieflydried under vacuum and dissolved in 2.8 mL of water and 1 mL of 0.1 NNaHCO₃ which had been sparged with helium. 6.7 mg of compound 3(IA-DABA-PEG) was added, and the mixture was kept for 16 hours at roomtemperature in the dark. The reaction mixture in a final volume of 6 mLwas applied to a 5×91 (1800 Ml) Pharmacia column which was packed withSephacryl 200 (Pharmacia). The column was eluted with 0.5 M NaCl, 0.1 Msodium borate, pH 8.3. A peristaltic pump was used and set to give aflow rate of approximately 2 mL per min., and fractions of 15 ml werecollected. The absorbance of the fractions at 260 nm was measured. Thefractions were also analyzed by polyacrylamide gel electrophoresis andthose containing pure conjugate were pooled.

Hybridization of Conjugate 3-I—Preparation of Conjugate 3-II

The pooled fractions from above contained 726 A₂₆₀ units. The equivalentamount of (TG)₁₀ was added and the tube was heated at 90° C. for tenminutes and then allowed to cool to room temperature over 1.5 hours. Anequal amount of isopropanol was added and the mixture kept for 3 hoursat −20° C. After centrifugation at 3000 rpm for 20 minutes, the pelletwas dissolved in 0.15 M NaCl, 0.01 M sodium citrate, pH 6.8. 53 mg ofthe hybrid was obtained. An aliquot of the material was diluted in theabove buffer and the melting temperature of the duplex was determined ina Carey 3E spectrophotometer. The material had a Tm of 73.4° C. and24.3% hyperchromicity. A 10 A₂₆₀ unit aliquot of the product wasannealed with excess (TG)₁₀ as described above. This as well asunannealed conjugate and a (TG)₁₀ standard were analyzed by gelpermeation HPLC on a Shodex Protein KW 8025 column on a Rainin HPLCinstrument. The column was eluted isocratically with 0.05M NaH₂PO₄, pH6.5, 0.5M NaCl. The run time was 12 minutes. The product had a retentiontime of 6.9 minutes and (TG)₁₀ 9.2 minutes. Comparison of the area underthe peaks showed that 98.09% of the product was double stranded DNA. Theconjugate is represented by the structure designated “Conjugate 3-II” inFIG. 6A.

EXAMPLE 6 Preparation of PN-KLH Conjugate

The PN-KLH conjugate was prepared according to the scheme below:

Synthesis of ACT-Modified (CA)₂₅

Compound 5 was coupled to (CA)₂₅ as the final step of automatedsynthesis. Forty-nine sequential steps were carried out usingalternating dC and dA phosphoramidites beginning with 10 g ofDMT-d-bzA-CPG support with a nucleoside loading of 30 μmol/g. The DMTrblocking group was removed from the resulting d-[DMTr-(bzCp(CE)bzA)₂₅],and 40 mL of activator solution (Milligen, Cat. No. MBS 5040) and 800 mgof compound 58 were added to the reaction mixture. The suspension wasmixed for 8 minutes by argon ebullition and subjected to a conventionaloxidation step. The support bound polynucleotide was removed from thereaction vessel, air dried, and treated with 100 mL of concentratedammonia for 40 hours at 55°. When cool, the mixture was filtered througha Gelman 10 μm polypropylene filter and the filtrate was then purifiedby conventional ion exchange chromatography. Fractions which absorbed at260 nm were further analyzed by polyacrylamide gel electrophoresis andthose containing pure product were combined and precipitated withisopropanol to provide 510 mg (31.9 μmol, 10%) of the ACT-modified(CA)₂₅.

Synthesis of Single-Stranded PN-KLH Conjugate

To a solution of 100 mg (2.5 μmol) of ACT-modified (CA)₂₅ in 1.33 mL of50 mM sodium borate pH 8.0 was added 31.3 mg (0.208 μmol) of KLH and 2.0mg (31.8 μmol) of NaCNBH₃. The mixture was kept at 37° C. for 72 h, andthe product was purified by chromatography on S-200.

Hybridization of Single-stranded PN-KLH Conjugate with (TG)₂₅

The equivalent amount of (TG)₂₅ was added to the single-stranded PN-KLHconjugate and the tube was heated at 90° C. for ten minutes and thenallowed to cool to room temperature over an hour and a half.Precipitation with isopropyl alcohol yielded 53 mg of 13; Tm (0.15 MNaCl, 0.01 M sodium citrate, pH 6.8) 73.4°, 31.1% hyperchromicity; 98%double stranded as determined by HPLC comparison to standards consistingof sample annealed with excess (TG)₁₀, unannealed conjugate, andunannealed (TG)₁₀ (Shodex Protein KW 8025 column, 0.05 N NaH₂PO₄, pH6.5, 0.5 M NaCl). This conjugate may be represented by the formulaKLH-[NH(CH₂)₅OPO₂.O-(CA)₂₅: (TG)₂₅]₋₅(assuming a molecular weight of 10⁵ for KLH) and is designated “PN-KLH.”Testing of Conjugate-3-II as a Tolerogen

Conjugate 3-II was tested for its ability to tolerize mice that had beenimmunized with an immunogenic form of the polynucleotide.

Material and Methods

Mice: C57BL/6 female mice 6 weeks of age were purchased from JacksonLaboratories, Bar Harbor, Me. The mice were housed and cared for by NIHapproved methods.

Immunization: The mice were primed, according to the method of Iverson(Assay for in vivo Adoptive Immune Response in Handbook of ExperimentalImmunology, Vol. 2 Cellular Immunology, Eds. D. M. Weir, L. A.Herzenberg, C. Blackwell and A. Herzenberg, 4th Edition, BlackwellScientific Publications, Oxford) by injecting the mice, i.p., with 100μg of PN-KLH precipitated on alum and with 2×10⁹ formalin fixedpertussis organisms as an adjuvant. The mice were boosted with 50 μg ofPN-KLH, in saline, i.p.

Coupling of PN to SRBC: Sheep Red Blood Cells (SRBC) in Alsevers werepurchased from Colorado Serum Co., Denver, Colo., and used within twoweeks. The SRBC were coated with (CA)₂₅:(TG)₂₅ (a 50 mer of CA:GT) bythe method of Kipp and Miller (“Preparation of Protein-Conjugated RedBlood Cells with ECDI (Modification)” in Selected Methods in CellularImmunology, (1980), Eds. B. B. Mishell and S. M. Shiigi, W. H. Freemenand Co., San Francisco, p. 103). Briefly, the SRBC were washed 4 timesin cold saline, mixed with 2 mg of (CA)₂₅:(TG)₂ coupled to D-EK in 0.35Mmannitol, 0.01 M Nacl containing 10 mg of carbodiimide and incubated for30 minutes at 4° C. The coated SRBC were washed twice with cold BalancedSalt Solution and resuspended to 10% (v/v).

Plaque assay: The number of anti-PN plaque forming cells (pfc) wasdetermined using the Cunningham technique (Marbrook, J., “Liquid Matrix(Slide Method)”, in Selected Methods in Cellular Immunology, (1980),Eds. B. B. Mishell and S. M. Shiigi, W. H. Freemen and Co., SanFrancisco, p. 86.) The number of IgG pfc were determined by eliminationof IgM plagues using rabbit and anti-mouse IgG as described by Henry(“Estimation of IgG responses by Elimination of IgM Plaques” in SelectedMethods in Cellular Immunology, (1980), Eds. B. B. Mishell and S. M.Shiigi, W. H. Freemen and Co., San Francisco, p. 91). Briefly, spleenswere harvested and single cell suspensions made in balanced saltsolution (BSS). Guinea pig serum was added to polynucleotide coated SRBCto give a final dilution of 1:9 guinea pig serum, and enough rabbitanti-mouse IgG was added to give a final dilution of 1:100 rabbitanti-mouse IgG. Equal volumes of the SRBC mixture and diluted spleencells were mixed in microtiter wells and transferred to Cunninghamchambers. Each spleen was tested individually and in triplicate. Theedges of the chambers were sealed with paraffin and the chambers wereincubated at 37° C. for 1 hour. The number of plaques were enumerated byviewing the chambers under an inverted microscope.

Results

Mice were primed with PN-KLH precipitated on alum with pertussis as anadjuvant (A&P) and seven weeks later divided into groups of 3 mice each.The mice were treated, i.p., with doubling dilutions of PN-DABA-PEG,Conjugate 3-II five days later all of the mice, including the control,were boosted with 50 μg of PN-KLH, in saline, i.p. Four days later, thespleens were harvested and the number of IgG pfc determined. As shown inTable 2, all doses of Conjugate 3-II tested showed a significantreduction in the number of pfc as compared to the control group. TABLE 2Tolerogenic Activity of Conjugate 3-II (PN-DABA-PEG) pfc/10⁶ Dose spleencells % Reduction (μg/mouse) Mean (S.D.) Mean None 12865 (2846)  62.5 2868 (6809) 77.7  125  3331 (939) 74.1  250  3044 (1929) 76.3  500 1809 (759) 85.9 1000  2814 (554) 78.1

EXAMPLE 7 Preparation and Testing of Conjugate 20-II

Conjugation of 5′ Modified (CA)₁₀ to Valency Platform Molecule20—Preparation of Single-Stranded Conjugate 20-I

969 μL (789 mg, 3.89 mmol) of tri-n-butylphosphine was-added to asolution of 918 mg (0.14 mol) of 5′-modified (CA)₁₀ in 30 mL of H₂Ounder argon atmosphere. The mixture was stirred for 1 hour and then 2.4mL of a 3M HaCl solution was added followed by 42 mL of isopropanolwhich had been sparged with helium to remove oxygen. The mixture wasplaced in a freezer at −20° C. for 1 hour and then-centrifuged at 3000rpm for 30 minutes. The supernatant was removed and the oily residue wasdissolved in 15.5 mL of helium sparged H₂O. 1.24 mL of 3M NaCl and 21.7mL of helium sparged isopropanol was added to the mixture. The resultingmixture was then placed in a freezer at −20° C. for 1 hour andcentrifuged at 3000 rpm for 20 minutes. The oily pellet was dried-undervacuum for 18 hours to yield a solid. The solid was dissolved in 6 mL ofhelium sparged H₂O to give a total volume of 6.4 mL. The amount of DNAwas 863 mg as determined by UVW absorbance at 260 nm (0.033 mg per unitof absorbance in pH 7.5 phosphate buffered saline). The solution wastransferred to a 50 mL three-neck flask under argon. One neck of theflask had an argon gas inlet while the other two necks were stoppered.The total volume was adjusted to 7.7 mL with H₂O and 0.87 mL of heliumsparged 1M sodium phosphate buffer, pH 7.8, and 0.97 mL of MeOH. 1.9 mL(33.63 mg, 0.025 mmol) of a 17.7 mg/mL solution of compound 20 in MeOHwas added to the mixture. The resulting mixture was stirred under argonfor 20 hours and then diluted to 100 mL with a solution comprising 0.1 MNaCl, 0.05 N sodium phosphate, pH 7.5, and 10% MeOH. Purification wasaccomplished by chromatography on Fractogel® (equilibration: 0.1 M NaCl,0.05 M sodium phosphate, pH 7.5, 10% MeOH: elution gradient 0.5 M NaCl,0.05 M sodium phosphate, pH 7.5, 10% MeOH to 0.8 NaCl, 0.05 M sodiumphosphate, pH 7.5, 10% MeOH). Fractions containing pure conjugate 20-Ias evidenced by HPLC and polyacrylamide gel electrophoresis werecollected in 232 mL of eluent. The product and salts were precipitatedby adding an equal volume of isopropanol and placing same in a freezerat −20° C. for 1 hour. Dialysis against H₂O (2×100 vol) gave 335 mg ofconjugate 20-I (32 mL of 10.47 mg/mL, 0.033 mg/absorbance unit at 260nm).

Annealing of Conjugate 20-I with (TG)₁₀ to Form Double-StrandedConjugate 20-II

150 mg (14.33 mL of 10.47 mg/mL based on 0.033 mg/absorbance unit at 260nm) of conjugate 20-I and 157.5 mg (1.50 mL of 104.6 mg/mL based on0.033 mg/absorbance unit at 260 nm) of (TG)₁₀ were placed into a 50 mL,polypropylene centrifuge tube. The concentration was adjusted to 15mg/mL by adding 2.0 mL of pH 7.2 10× PBS and 2.17 mL of H₂O. The mixturewas placed in a 90° C. water bath and allowed to cool to roomtemperature over 1.5 hours. The concentration was determined to be 17.7mg/mL by absorbance at 260 nm (0.050 mg/absorbance unit); transitionmelt temperature 67.5° C.; hyperchromicity 27%; osmolality 346; pH 7.2.For final formulation of conjugate 20-II, the solution was diluted to afinal concentration of 12.7 mg/mL and an osmolality of 299 by adding7.23 mL of pH 7.2½× PBS and filtering through a 0.22μ filter.

Alternative Conjugation of 5′-Modified (CA)₁₀₋₂₀, Preparation of SingleStranded Conjugate 20-I

10 equivalents of tri-n-buxtylphosphine are added to a 10 mg/mL solutionof 5′-modified (CA)₁₀ in He sparged with 100 mM pH 5 sodium acetate. Themixture is stirred for 1 hour and then precipitated with 1.4 volumes ofisopropyl alcohol (IPA). The mixture is placed in the freezer at −20° C.for 1 hour and centrifuged at 3000 rpm for 20 minutes. The supernatantis removed and the pellet is dissolved to 10 mg/mL in He sparged IPA.The mixture is placed in the freezer at −20° C. for 1 hour andcentrifuged at 3000 rpm for 20 minutes. The pellet is dried under vacuumfor 18 hours to give a solid. A 50′ mg/mL solution of the solid isprepared in He sparged 100 mM pH 10 sodium borate buffer. 0.25equivalents of compound 20 as a 40 mg/mL solution in 9/1 M OH/H₂O isadded to the mixture. The mixture is stirred at room temperature for3-20 hours and diluted (0.1 M NaCl, 0.05 sodium phosphate, pH 7.5, 10%MeOH). Purification is accomplished by chromatography on Factogel(equilibration; 0.1 M NaCl, 0.05 N sodium phosphate, pH 7.5, 10% MeOH:elution gradient; 0.5 M NaCl, 0.05 M sodium phosphate, pH 7.5, 10% MeOHto 0.8M NaCl, 0.05 sodium phosphate, pH 7.5, 10% MeOH). Fractionscontaining pure 2, as evidenced by HPLC and, polyacrylamide gelelectrophoresis, were collected. The product and salts are precipitatedby adding an equal volume of IPA and standing in the freezer at −20° C.for 1 hour. Dialysis against H₂O (2×10 vol) give 20-I.

Alternative Annealing of 20-I with (TG)₁₀₋₂₀ to Form Double StrandedConjugate 20-II

The methodology is essentially the same as that described above exceptthat annealing is done at 70° C. instead of 90° C.

Second Alternative Conjugation of 5′-Modified (CA)₁₀₋₂₀, Preparation ofSingle Stranded Conjugate

4.8 mL of tri-n-butylphosphine was added to a solution of 7.75 g of5′-modified. (CA)₁₀ in 104 mL of Ar sparged 100 mM pH 5 sodium acetateunder N₂. The mixture was stirred for 1 hour and then precipitated with232.5 mL of IPA. The mixture was placed in a freezer for −20° C. for 1.5hours, centrifuged at 3000 rpm for 20 minutes and then frozen at −20° C.for 24 hours. The supernatant was removed and the pellet was dissolvedin 170 mL He sparged 0.3 M NaCl solution. The mixture was againprecipitated with 232 mL of Ar sparged IPA. The mixture was then placedin a freezer at −20° C. for 2 hours, centrifuged at 3000 rpm for 20minutes and then from at −20° C. for 11 hours. The supernatant wasdecanted and the pellet was dried under vacuum for 12 hours to give asolid. A solution of the solid was prepared in 110 mL of Ar sparged 100mM pH 10 sodium borate buffer. 406 mg of compound 20 as a solution in4.4 mL of 9/1 MeOH/H₂O was added to the mixture. The mixture was stirredat room temperature for 2 hours. The product mixture contained 62% of20-I by high-pressure ion chromatography, Waters Gen Pak Fax column(100×4 mm), 60° C., linear gradient from 65% A/35% B to 18% A/82% B;A=0.05 M NaH₂PO₄, pH 7.5, 1 mM EDTA, 10% MeOH (v/v); B=0.05 M NaH₂PO₄,pH 7.5, 1 M NaCl, 1 mM EDTA, 10% MeOH (v/v), eluting at 19.5 minutes.

Testing of Conjugate 20-II and Nonconjugated Controls

C57BL/6 mice-were immunized with PN-KLH and A&P. After three weeks,groups of 5 mice/group were treated with either different doses ofConjugate 20-II or 4.5 hM HAD-AHAB-TEG (linker, HAD, attached toderivatized valency platform molecule, AHAB-TEG, see FIG. 7), or 18 nM(4×4.5) (CA)₁₀: (TG)₁₀, or a mixture of 4.5 nM HAD-AHAB-TEG plus 18 nM(CA)₁₀:(TG)₁₀, i.p.; and one group was not treated. The groups weregiven booster injections and the sera were collected and assayed asdescribed in Example 6. The percent reduction of the anti-PN response isshown in FIG. 4. The anti-KLH responses of these mice was normal andwere not significantly different than those shown in FIG. 2. The resultsclearly show that the anti-PN response was not affected by (i) thevalency platform molecule alone, (ii) the PN alone, or (iii) a mixtureof the two. The PN must be coupled to the nonimmunogenic valencyplatform molecule in order to induce tolerance.

Conjugate 20-II Causes a Reduction in the Number of PN-Specific AntibodyProducing Cells

C57Bl/6 mice were immunized with PN-KLH, A&P. After three weeks, groupsof 3 mice/group were treated with different doses of Conjugate 20-II,i.p; one group was not treated. After five days, all of the mice weregiven a booster injection of PN-KLH in saline, i.p., and then 4 dayslater their spleens were harvested and assayed for the number ofPN-specific, IgG-producing cells using the hemolytic plaque assay. Theresults, shown in Table 3, clearly show that this conjugate reduced thenumber of PN-specific IgG-producing cells. TABLE 3 REDUCTION IN THENUMBER OF pfc BY Conjugate 20-II PN-specific pfc per 10⁶ Dose spleencells Group# μg/mouse (Mean & S.E.) % Reduction 1 None 5562 (2570) 2 274 982 (1871) 82.3 3 91 1867 (1335) 66.4 4 30 2247 (1606) 59.6 5 10 6109(2545) 0 6 3 4045 (1411) 27.3 7 1 4578 (2475) 17.7 8 0.4 5930 (897) 0

EXAMPLE 8 Testing of Conjugates as Tolerogens

Testing of Conjugate 17-II as a Tolerogen

C57BL/6 mice were immunized with PN-KUA, A&P. Three weeks later groupsof 5 mice/group were treated with different doses of Conjugate 17-IIintraperitoneally, (i.p.), and one group was not treated. Five dayslater all of the mice were given a booster injection of PN-KLH, insaline, i.p., and 7 days later the mice were bled. The sera wereanalyzed for anti-PN antibody by the Farr assay at a PN concentration of10⁻⁸M. The percentage reduction of the anti-PN response is shown inFIG. 1. The sera were also analyzed for anti-KLH antibodies using anELISA assay. The results, expressed as the percentage of anti-KLHcompared to a standard pool of anti-KLH sera are shown in FIG. 2. Thedata in FIG. 1 show that this-conjugate reduces the anti-PN response.The anti-KLH (platform molecule) response in all of the mice is normal(see FIG. 2).

Testing of Various of the 11 Series of Conjugates as Tolerogens

Groups of three C57BL/6 mice/group were immunized with PN-KLH, A&P.After three weeks, two groups were treated with 3 different doses ofeither Conjugate 11-IV, Conjugate 11-II, Conjugate 11-VI or Conjugate11-VIII, i.p., one group was not treated. These conjugates are describedin FIGS. 6A-B and were prepared according to the methodology describedabove in Example 7. Five days later all of the mice were given a boosterinjection of PN-KLH, in saline, i.p., and 7 days later the mice werebled. The sera were analyzed for anti-PN antibody by the Farr assay at aPN concentration of 10⁻⁸M. The results showing the percentage reductionin th anti-PN response are-presented in FIG. 3. The anti-KLH responsesin these mice were not significantly different than the responses shownin FIG. 2. All four conjugates significantly reduced the anti-PNresponse at all doses tested.

Conjugate 11-II Causes a Reduction in the Number of PN-Specific AntibodyProducing Cells

C57Bl/6 mice were immunized with PN-KLH, A&P. After three weeks, groupsof 3 mice/group were treated with different doses of Conjugate 11-II,i.p., one group was not treated. Five days later, all of the mice weregiven booster injections of PN-KLH in saline, i.p.; and 4 days latertheir spleens were harvested and assayed for the number of PN-specific,IgG-producing cells using the hemolytic plaque assay. The results ofthis experiment with different doses of Conjugate 11-II are shown inTable 4. These results clearly show that this conjugate reduced thenumber of PN-specific IgG-producing and that the reduction in antibodytiter was not due to the clearance of serum antibody bound to conjugate.TABLE 4 REDUCTION IN THE NUMBER OF pfc BY Conjugate 11-II PN-specificpfc per 10⁶ spleen cells % Reduction Group# μg/mouse (Mean & S.E.) (SD)1 None 10845 (1308) 2 263  3613 (547) 66.23 (8.6) 3 87  3462 (1041)64.98 (17) 4 29  7354 (1504)  29.5 (23.8) 5 9  6845 (2031)  30.9 (32.2)6 3  7982 (223)  26.8 (3.52) 7 1  6043 (545)  44.5 (7) 8 0.4  9343(1251)   13 (19.8)

EXAMPLE 9 Preparation of HADps-(CA)₁₀—Conjugate 20-IV

A modified polynucleotide with a phosphorothioate joining the linker tothe 5′ end was prepared. Synthesis of the twentymer, (CA)₁₀, and theaddition of the HAD linker to the polynucleotide was carried outaccording to the methodology of Example 5 except for the following. Inthe final oxidation step, the iodine solution was replaced with a 0.05 Msolution of 3H-1,2-benzodithiole-3-one 1,1 dioxide (Glen Research,Sterling, Va.) in acetonitrile. Sulfurization was carried out accordingto the manufacturer's instruction. Ammonia treatment and purificationwere carried out as in Example 5. Conjugation of the polynucleotide tothe AHAB-TEG valency platform were carried out according to themethodology of Example 5.

Testing of Conjugate 20-IV as a Tolerogen

Because the 5′ phosphate on the PN may be susceptible to enzymaticdegradation, one of the oxygen molecules on the terminal phosphate wasreplaced with sulfur—thus the name HAD_(P)S. C57BL/6 mice were immunizedwith PN-KLH, A&P. After three weeks, groups of 5 mice/group were treatedwith different doses of Conjugate 20′-IV, i.p.; one group was nottreated. The groups were given booster injections and the sera werecollected and assayed as described above. The results showing thepercentage reduction in the anti-PN response are shown in FIG. 5. Theanti-KLH responses of these mice (data not shown) were normal and werenot significantly different that those shown in FIG. 2. These resultsshow that this conjugate significantly reduced the anti-PN response.

Conjugate 20-IV Causes a Reduction in the Number of PN-Specific AntibodyProducing Cells

C57Bl/6 mice were immunized with PN-KLH, A&P. After three weeks, groupsof 3 mice/group were treated with different doses of Conjugate 20-IV,i.p.; one group was not treated. After five days, all of the mice weregiven a booster injection of PN-KLH in saline, i.p., and 4 days latertheir spleens were harvested and assayed for the number of PN-specific,IgG-producing cells using the hemolytic plaque assay. The results, shownin Table 5, show that this conjugate reduced the number of PN-specificIgG-producing cells. TABLE 5 REDUCTION IN THE NUMBER OF pfc BY Conjugate20-IV PN-specific pfc per 10⁶ Dose spleen cells Group# μg/mouse (Mean &S.E.) % Reduction 1 None 5889.4 (3444) 2 274   3413 (1604) 42 3 91   222(752) 96.2 4 30   1492 (2269) 74.7 5 10   5421 (832) 8 6 3   5077 (1946)13.9 7 1   7023 (679) 0 8 0.4   4159 (2688) 29

EXAMPLE 10 Treatment of BXSB Mice With LJP, 394, Conjugate 20-II

Mice Treatment Protocol

Six to 9 week old male BXSB mice (Jackson Laboratory, Bar Harbor, Me.)were housed at the La Jolla. Pharmaceutical facility. Food and waterwere provided as libitum. Animals were rested one week prior to use.Initial blood samples and weights were obtained prior to the firstconjugate treatment. Conjugate treatment was initiated at 7 to 9 weeksof age and was administered intravenously twice weekly from day 59 today 150. Animals were bled periodically and their anti-DNA antibodytiters were determined.

Assay for IgG Anti-DNA Antibody Production

A serum sample taken from each mouse was assessed for the presence ofanti-DNA antibody by ELISA. Falcon Probind 96 well microtitration assayplates (Becton Dickerson, Oxnard, Calif.) were coated with 100 μL/wellof (PN)₅₀-D-EK (a co-polymer of D-glutamic acid and D-lysine) at aconcentration of 50 μg/mL overnight at 4° C. The plates were washedtwice with PBS without calcium and magnesium and 40.05% Tween 20 (washbuffer) using a M96V plate washer (ICN Biomedical, Inc., Irvine,Calif.). Plates were blocked for 1 hour at room temperature in PBScontaining 1% gelatin (Norland Products, Inc., New Brunswick, N.J.) and0.05% Tween 20. Plates were washed twice with wash buffer before theaddition of serum samples or standards. Serum samples and standards wereprepared in a diluent containing PBS with 1% gelatin, 0.05% Tween 20 and10% goat serum. Plates were incubated with serum samples for 60 to 90minutes at 37° C. and then the wells were washed four times with washbuffer. Biotinylated goat anti-mouse IgG (Sigma Chemical Co., St. Louis,Mo.) was diluted 1/1000 in blocking solution containing 10% goat serum.The plates were incubated for 1 hour at 37° C. and washed four times.The substrate, OPD (Sigma Chemical Co., St. Louis, Mo.) was added. Theplates were incubated in the dark until the highest reading of thehighest standard was approximately 1 OD unit by an ELISA plate reader atOD 450 nm (Bio-Tek Instruments, Winooski, Vt.). The reaction was stoppedwith 50 μL of 3M HCl and the plates were read at 490 nm. The referencepositive serum was included in each microtitration plate and thepositive wells from each assay were within the sensitivity range of thereference curve 95% of the time. In the later bleeds, some positivesamples exceeded the reference curve. However the most dilute mouseserum sample was within the range of the reference curve. No significantbinding was observed by normal control negative serum.

EXAMPLE 11 Preparation of Melittin Peptides and Conjugate

The melittin molecule, composed of 26 amino acids, is one of the majorcomponents of bee venom. One third of the bee venom sensitiveindividuals have melittin specific antibodies. Melittin is highlyimmunogenic in some mouse strains (Balb/c, CAF1). The majority (>80S %)of melittin specific antibodies in the responder mouse strains bind a Bcell epitope which is the c-terminal heptapeptide of melittin. MelittinH₂N-Gly-Ile-Gly-Ala-Val-Leu-Lys- (SEQ. ID.: 1)Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala- Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-CONH_(2.) Melittin Peptides for T cell Stimulation MelittinPeptide #1. Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly (SEQ. ID NO.: 2) (“7 mer”).Melittin Peptide #2. Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln- (SEQ. ID NO.: 3)Gly (“8 mer”). Melittin Peptide #3. Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-(SEQ ID NO.: 4) Gln-Gly (“9 mer”). Melittin Peptide #4.Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg- (SEQ. ID NO.: 5) Gln-Gln-Gly (“10mer”). Melittin Peptide #5. Cys-Ile-Ser-Trp-Ile-Lys-Arg-Lys- (SEQ. IDNO.: 6) Arg-Gln-Gln-Gly (“11 mer”).Peptide Synthesis

Melittin peptides were synthesized-using standard Fmoc chemistrytechniques on a glycine resin (Advanced ChemTech #SG5130 or equivalent(Advanced ChemTech, 2500 Seventh Street Road, Louisville, Ky.) using 2.3M excess amino acid derivatives for each coupling step. Completion ofthe coupling was monitored with bromphenol blue and confirmed withninhydrin. Melittin Peptides Used in Conjugations Melittin Peptide #6.H₂N-Cys-Trp-Ile-Lys-Arg-Lys-Arg- (SEQ. ID NO.: 7) Gln-Gln-Gly-CO₂H.Melittin Peptide #7. H₂N-Trp-Ile-Lys-Arg-Lys-Arg-Gln- (SEQ. ID NO.: 8)Gln-Lys-Cys-Gly-CO₂H. Melittin Peptide #8.H₂N-Cys-Ile-Ser-Trp-Ile-Lys-Arg- (SEQ. ID NO.: 9)Lys-Arg-Gln-Gln-Gly-CO₂H. Melittin Peptide #9.(H₂N-Trp-Ile-Lys-Arg-Lys-Arg-Gln- (SEQ. ID NO.: 10)Gln)₂-Lys-Cys-Gly-CO₂H.

A cysteine was added as required for coupling certain peptides via athioether bond to the valency platform molecule. Peptides were purifiedby reversed phase HPLC following synthesis and lyophilized to dryness.The appropriate amount of peptide was then weighed out for eachconjugation.

Reduction of Preformed Disulfide Bonds: (Tributylphosphine Method)

All buffers were sparged with helium. The peptide was dissolved in aminimal volume (approximately 10 to 20 mg/mL) of 0.05 M NaHCO₃ (pH8.25). A 1 mL solution of 0.7 M tributylphosphine (TBP; MW 202.32g/mole; d-0.812 g/mL) was prepared by adding 174.4 μL of TBP to 825.6 μLof isopropanol (iPrOH). Then, 1:1 equivalents of TBP were added to thepeptide solution prepared as described above, mixed well, and allowed toreact for 30 minutes to 1 hour with occasional mixing to keep TBPdissolved and/or dispersed in the solution. Complete reduction wasconfirmed by HPLC.

Conjugation of Peptides to Valency Platform Molecule #3 of #60:

All buffers were sparged with helium. The polyethylene glycol (PEG)derivative #3-or #60 was dissolved in a minimal volume (approximately 20mg/mL) of 0.05 M NaHCO₃ (pH 8.25). Approximately 3 equivalents ofpeptide were used per iodacetyl group on the PEG derivative. Forpara-aminobenzoic acid (PABA)-PEG; 2 iodacetyl groups; MW=approximately4100 g/mole; 6 equivalents of peptide were used for each equivalent ofPABA-PEG. For diaminobenzoic acid (DABA)-PEG; 4 iodoacetyl groups;MW=approximately 4300 g/mole; 12 equivalents of peptide were used foreach equivalent of: DABA-PEG. The PEG solution was added to the reducedpeptide solution and allowed to react for at least one hour in the dark.The peptide conjugate was purified by preparative HPLC. Before poolingand lyophilization, fractions were checked by electrophoresis using a15% tricine gel. TABLE 6 Conjugates of melittin Peptides and PEG T cell# B cell Conju- activation by Conjugate Valence Peptide epitopes/ gationpeptide or number platform conjugated molecule terminus conjugate¹ 1 5 62 N no(pep) 2 3 6 4 N no(pep/conj) 3 3 7 4 C nd 4 3 5 4 N yes(pep) 5 3 8 8² C nd¹Stimulation of uptake of [³H] thymidine by cultured T cell frommelittin-immunized mice; nd = not determined; pep = peptide tested;.conj = peptide-PEG conjugate tested.²4 copies of a branched peptide, containing two identical branches each;each branch comprising a B cell epitopeMurine Lymph Node Proliferation Assays.

Femal Balb/c mic (6-8 weeks old; Jackson Laboratory, Bar Harbor, Me.)were obtained and housed at the La Jolla Pharmaceutical animal facilityaccording to National Institutes of Health guidelines. Food and waterwas provided ad libitum. Balb/c mice were immunized in each hind footpadwith 50 μg of melittin molecule in Complete Freund's Adjuvant (CFA)(Sigma Chemical Co., St. Louis, Mo.). Popliteal lymph nodes wereharvested aseptically seven days later. Lymph nodes were gentlydissociated by teasing the cells through a 50 mesh sieve screen. Thesingle cell suspension was washed in RPMI-1640 (Irvine Scientific,Irvine Calif.) containing glutamine, penicillin and streptomycin. 5×10⁵cells in RPMI medium supplemented with 10% fetal bovine serum (FCS) inquadruplicate wells of round bottom 96-well Corning microtitrationplates were-cultured with melittin or a melittin peptide at 10, 1.0 or0.1 μg/mL. Cells in the positive control wells were cultured with murineinterleukin 2 (IL-2) at 100 or 50 U/mL, PHA (phytohemagglutinin) at 1μg/mL. The negative control wells contained lymph node cells in RPM-1640and 10% FCS. The cells were cultured for 4 days in a 37° C. incubatorwith 5% CO₂. Each well was pulsed with 1 μCi of [³H]thymidine (ICNBiochemicals, Costa Mesa, Calif.) for an additional 18 hours. Cells wereharvested onto a glass fiber filter mat using a semiautomatic cellharvester (Scatron, Sterling, Va.). Incorporation of [³H]thymidine wasdetermined by liquid scintillation. The results were expressed asaverage counts per minute.

In Vivo Protocols

Balb/c mice were primed intraperitoneally (i.p.) with 4 μg of melittinin CFA. One month later the potential tolerogen or formulation bufferwas administered i.p. Three days later all mice received an i.p.injection of 4 μg of melittin in Incomplete Freund's Adjuvant (ICF)(Sigma Chemical Co., St. Louis, Mo.). 100 to 200 μL of blood wascollected from the retro-orbital venous plexus 10 days later. Serumsamples were assayed for anti-peptide or anti-melittin IgG antibodies.

Assay for IgG Anti-Melittin or Total Anti-Melittin Antibodies

An individual mouse's serum sample was assessed serially for thepresence of anti-melittin antibodies by ELISA. Falcon Probind 96-wellmicrotitration plates were: precoated with 10 μg/mL melittin or melittinpeptide in phosphate buffered saline (PBS), pH 7.2, overnight at 4°. Theplates were washed twice with a wash solution containing PBS, 0.02%Tween 20, and 1% gelatin (Norland Products Inc., New Brunswick, N.J.).Plates were blocked with 200 μL PBS containing 5% gelatin for 1 hour at37°. Serum samples were prepared in a diluent of PBS containing 5%gelatin. Samples were tested at dilutions of 1:100 to 1:1000. After 1hour of incubation at 37° C., the plates were washed four times.ExtraAvidin peroxidase (Sigma Chemical Co., St. Louis, Mo.) was diluted1:1000 in PBS containing 5% gelatin. The plates were incubated 30minutes at 37° C. and then washed five times. Wells were developed witho-phenylenediamine (OPD) (Sigma Chemical Co., St. Louis, Mo.) in thedark for 15-30 minutes, the reaction was stopped with 3 M HCl. Theoptical density (OD) was determined at 450 nm on a microplate reader(Bio-tek Instruments, Winooski, Vt.).

Antibody Forming Cell Assay

Cellulose microtitration plates (Millipore Co., Bedford, Mass.) wereprepared as indicated above for the IgG antibody (ELISA) assay. However,at the point in the assay where the serum samples were added to thewells, splenic cells (5×10⁵/well) were added instead of serum, andincubated overnight. The remainder of the ELISA assay was performed asindicated above.

T Cell Epitopes

T Cells from mice primed with melittin showed T cell proliferation inresponse to the whole melittin molecule and to C-terminal melittinpeptides 3, 4, and 5 (FIG. 8). However, C-terminal peptides 1 and 2induced no significant T cell proliferation. Melittin peptides 2 and 5were conjugated to PEG. Like melittin peptide 2, the PEG conjugate ofmelittin peptide 2 (Conjugate 2) also did not induce significant T cellproliferation.

Studies Using Melittin Conjugated Peptides to Tolerize Mice Primed andBoosted With Melittin

Mice treated with Conjugate 2 (10 mg/kg, 200 μg/mouse), hadsignificantly lower levels of anti-melittin peptide 2 antibodies (FIG.9) and also lower levels of anti-melittin antibodies (FIG. 10) ascompared to the control Balb/c mice treated with formulation buffer.Spleen cells from mice treated with buffer control or Conjugate 2 wereassayed for the ability of antibody forming cells to produceanti-melittin or anti-melittin peptide 2 antibodies as measured in asoluble ELISA assay. As shown in FIG. 11, the levels of anti-melittinpeptide 2 antibody forming cells in the Conjugate 2 treatment group weresignificantly lower than in the control group which was administeredformulation buffer. Mice treated with Conjugate 4, a conjugate ofpeptide 5 which contains a T cell epitope, failed to reduce the titer ofantibodies to peptide 5 in treated mice. Thus, the conjugate containinga T cell epitope was not a tolerogen (FIG. 12). In fact, rather thanreduce the response, the levels of anti-peptide antibody may haveincreased slightly.

EXAMPLE 12 Additional Studies Using Melittin Peptide Conjugates toTolerize Mice Primed and Boosted With Melittin

Female C57BL/6 mice, ages 5 to 8 weeks were purchased from The JacksonLaboratory, Bar Harbor, Me. Animals were maintained and treatedaccordingly to National Institutes of Health guidelines.

Immunization Protocol

Mice were primed by an i.p. injection containing 5 μg of melittinprecipitated on alum and 2×10⁹ B. pertussis (Michigan Department ofPublic Health, Lansing, Mich.) as an adjuvant. The mice were boostedwith 5 μg of melittin, i.p., in PBS.

pfc Assay

Sheep Red Blood Cells (SRBC) (Colorado Serum Co., Denver, Colo.) wereconjugated with melittin-peptide 2 using carbodiimide. Fresh SRBC (lessthan 2 weeks old) were washed four times with cold saline and one timewith mannitol (0.35 M mannitol, 0.01 M NaCl). The SRBC were suspended inmannitol to a concentration of 10% (v/v). 100 μL of mannitol containing30 μg of melittin peptide #3 were added to 1 mL aliquots of 10% SRBCwhich were then incubated on ice for 10 minutes. 100 μL of a 100 mg/mLsolution of 1-ethyl-3 (3-dimethylaminopropyl)-carbodiimide HCl (EDCI)was then added and incubated on ice for 30 minutes. The SRBC were washedtwice with Balanced Salt Solution (BSS) (Irvine Scientific Co, Irvine,Calif.) and resuspended to 10% (v/v). Lyophilized guinea pig complement(GIBCO, New York, N.Y.) was reconstituted with BSS and then diluted 1:3with BSS. One mL of the diluted guinea pig complement was added to 3 mLof conjugated SRBC. Rabbit anti-mouse IgG was added to give a finaldilution of 1:100 of the rabbit antiserum. This concentration waspredetermined to inhibit all IgM pfc while enhancing the maximum numberof IgG pfc. An equal volume of this complement/anti-mouse IgG/SRBCsuspension was mixed with a cell suspension of mouse spleen cells takenfrom a single mouse. 50 μL of each mixture was transferred to thechambers of a Cunningham slide (three chambers per slide). The edgeswere then sealed with paraffin and incubated at 37° C. for one hour. Thenumber of plaques per chamber was counted with the aid of a dissectingmicroscope. Each spleen suspension was also assayed using non-conjugatedSRBC as a control. The number of viable cells, in each spleen cellsuspension, was determined. The number of pfc per 10⁶ spleen cells wasdetermined for each chamber and the mean of the triplicates calculated.The number of pfc for non-conjugated SRBC was subtracted from the numberof pfc for conjugated SRBC to determine the number of peptide-specificpfc.

Determining The Optimal Time to Measure pfc

Mice were primed with melittin. Groups (3 mice per group) of primed micewere boosted with melittin on days 2, 4, 6, and 8. On day 10 the micewere sacrificed and their spleens harvested. Cell suspensions wereprepared and assayed for the number of peptide specific pfc determined.The optimal number of pfc was obtained 6 days after boosting withmelittin.

The Orientation of the Peptide on the PEG Conjugate Does Not Affect theConjugate's Ability to Induce Tolerance

Two different tolerogens were constructed to determine if theorientation of the peptide on the PEG conjugate affects its ability toinduce tolerance. The peptide was covalently bound to valency platformmolecule 3 through its C-terminal end to make melittin conjugate 3.Groups (3/group) of mice primed with melittin were treated, i.p., withconjugates or with saline. Five days later all of the mice, includingthe non-treated control group, were boosted with 5 μp of melittin. Sixdays later the mice were sacrificed, their spleens were harvested andthe number of peptide specific pfc determined. As-illustrated in Table6, both orientations were effective in reducing the number ofpeptide-specific pfc/10⁶ spleen cells in mice primed and boosted withthe parent protein Melittin. TABLE 7 Orientation of the peptide on thePEG conjugate does not affect the conjugates' ability to inducetolerance Peptide specific Melittin pfc per 10⁶ spleen Conjugate#μg/mouse cells (Mean and S.D.) % Reduction 3 1000 μg   386 (85) 86.8% ″ 500 μg   489 (one mouse) 83.3% ″  250 μg   957 (298) 67.3% 2 1000 μg  546 (160) 81.3% ″  500 μg 866.6 (235) 70.4% ″  250 μg  1280 (onemouse) 56.2% None None  2924 (164) —The Number of Peptides per PEG Conjugate Does Affect the Conjugate'sAbility to Induce Tolerance

Three different conjugates, each with a different number of peptides perPEG conjugate, were constructed to determine if the ratio of peptides toPEG molecule was important. Conjugate 1 had only two peptides per PEGconjugate. Another had four peptides per PEG conjugate (Conjugate 2).The third had eight peptides per PEG conjugate (Conjugate 5). Groups(3/group) of mice primed with melittin were treated, i.p., with thedifferent conjugates or with saline. Five days later all of the nice,including the non-treated control group, were boosted with 5 μg ofmelittin. Six days later, the mice were sacrificed, their spleens wereharvested and the number of peptide-specific pfc determined. As shown inTable 8, Conjugate 1, containing two peptides per PEG molecule, wasineffective in reducing the number of peptide-specific pfc/10⁶ spleencells in mice primed and boosted with the parent protein melittin. Theresults show that both melittin conjugates 2 and 5 were effective astolerogens; however, conjugate 5, which contained 8 peptides, waseffective at a lower dose than conjugate 2 which contained four peptidesper valency platform molecule. TABLE 8 The number of peptides per PEGconjugate does affect the conjugates' ability to induce tolerancePeptide specific indirect Treatment Dose IgG Molecule μg/mouse (nMoles)pfc(SD) % Reduction No treatment 1159 (280) std Conjugate 1 1000 (217)1290 (98)  −11%  250 (54) 1350 (206)  −16% Conjugate 2  500 (80)  585(125)  49.5%  250 (40) 1001 (176)    14% Conjugate 5  500 (53)  630(325)  45.6%  250 (26.5)  443 (105)  61.8%  125 (13.25)  583 (69)  49.7%

Modifications of the above-described modes for carrying out theinvention that are obvious to those of skill in the fields ofpolynucleotide chemistry, conjugation chemistry, immunology and relatedfields are intended to be within the scope of the following claims.

1. A conjugate of at least one biological molecule and a valencyplatform, said valency platform having the formula:Q₁-(CH₂CH₂O)_(n)CH₂CH₂-Q₁ wherein each Q₁ is a chemical moietyindependently selected from the group consisting of:

wherein each Q₂ is a chemical moiety independently selected from thegroup consisting of:

wherein each Q₃ is a chemical moiety independently selected from thegroup consisting of:

wherein each T is a chemical moiety independently selected from thegroup consisting of:

wherein: n is an integer from 0 to 300; m is in integer from 1 to 10; pis an integer from 1 to 10; each X is independently F, Cl, Br, I, orother good leaving group; each R^(ALK) is independently a linear,branched, or cyclic alkyl group of 1 to 20 carbon atoms; each R^(SUB) isindependently H, a linear, branched, or cyclic alkyl group of 1 to 20carbon atoms, an aryl group of 6 to 20 carbon atoms, or an alkaryl groupof 7 to 30 carbon atoms; each R^(ESTER) is independently N-succinimidyl,p-nitrophenyl, pentafluorophenyl, tetrafluorophenyl,2,4,5-trichlorophenyl, 2,4-dinitrophenyl, cyanomethyl, or otheractivating group; and each R^(B) is independently a chemical moietycomprising 1 to 50 atoms selected from the group consisting of C, H, N,O, Si, P, and S.
 2. A conjugate according to claim 1, wherein each Q₂is:


3. A conjugate according to claim 2, wherein each Q₁ is:


4. A conjugate according to claim 2, wherein each Q₁ is:


5. A conjugate according to claim 2, wherein each Q₁ is:


6. A conjugate according to any one of claims 3 to 5, wherein each Q₃ isa chemical moiety independently selected from the group consisting of:


7. A conjugate according to any one of claims 3 to 5, wherein each Q₃ isa chemical moiety independently selected from the group consisting of:


8. A conjugate according to claim 6, wherein each T is a chemical moietyindependently selected from the group consisting of:


9. A conjugate according to claim 7, wherein each T is a chemical moietyindependently selected from the group consisting of:


10. A conjugate according to any one of claims 1 to 5, wherein each saidbiological molecule is selected from the group consisting of acarbohydrate, a lipid, a lipopolysaccharide, a peptide, a protein, aglycoprotein, a single stranded oligonucleotide, a double-strandedoligonucleotide, a hapten, and an analog thereof or a combinationthereof.
 11. A conjugate according to claim 8, wherein each saidbiological molecule is selected from the group consisting of acarbohydrate, a lipid, a lipopolysaccharide, a peptide, a protein, aglycoprotein, a single stranded oligonucleotide, a double-strandedoligonucleotide, a hapten, and an analog thereof or a combinationthereof.
 12. A conjugate according to claim 9, wherein each saidbiological molecule is selected from the group consisting of acarbohydrate, a lipid, a lipopolysaccharide, a peptide, a protein, aglycoprotein, a single stranded oligonucleotide, a double-strandedoligonucleotide, a hapten, and an analog thereof or a combinationthereof.
 13. A conjugate according to any one of claims 1 to 5, whereineach said biological molecule is an analog of an immunogen wherein (a)said analog binds specifically to B cells to which said immunogen bindsspecifically and (b) said conjugate lacks a T cell epitope.
 14. Aconjugate according to claim 8, wherein each said biological molecule isan analog of an immunogen wherein (a) said analog binds specifically toB cells to which said immunogen binds specifically and (b) saidconjugate lacks a T cell epitope.
 15. A conjugate according to claim 7,wherein each said biological molecule is an analog of an immunogenwherein (a) said analog binds specifically to B cells to which saidimmunogen binds specifically and (b) said conjugate lacks a T cellepitope.
 16. A conjugate according to any one of claims 1 to 5,comprising a plurality of biological molecules, wherein each of saidbiological molecules comprises a polynucleotide duplex, said duplexhaving a significant binding activity for human systemic lupuserythematosus anti-double stranded DNA autoantibodies.
 17. A conjugateaccording to claim 16, wherein each of said duplexes are coupled to saidvalency platform via a linker group.
 18. A conjugate according to claim16, wherein each of said duplexes are coupled to said valency platformvia a linker group derived from a thio-6 carbon chain phosphate or athio-6 carbon chain phosphorothioate.
 19. A conjugate according to claim16, wherein said duplexes are substantially homogeneous in length.
 20. Aconjugate according to claim 16, wherein said duplexes are substantiallyhomogeneous in nucleotide composition.
 21. A conjugate according toclaim 16, wherein said duplexes are 20 to 50 base pairs in length.
 22. Aconjugate according to claim 16, wherein said duplexes are covalentlybonded to said valency platform at or proximate to one of their ends.23. A conjugate according to claim 16, wherein said conjugate suppressesantibody production associated with human systemic lupus erythematosus.24. A conjugate according to claim 16, wherein said duplexes have aB-DNA helical structure and a significant binding activity for humansystemic lupus erythematosus anti-double stranded DNA autoantibodies.25. A composition comprising a conjugate according to claim 16formulated with a pharmaceutically acceptable injectable vehicle.
 26. Acomposition comprising a conjugate according to claim 13 formulated witha pharmaceutically acceptable injectable vehicle.
 27. A method ofsuppressing antibody production in an individual in need thereofcomprising administering the composition according to claim 43 to theindividual in an effective amount such that antibody production issuppressed.
 28. A method of suppressing antibody production in anindividual in need thereof, said method comprising administering thecomposition according to claim 43 to the individual in an effectiveamount such that antibody production contributing to thyroiditis,stroke, or myasthenia gravis is suppressed.
 29. A method of suppressingantibody production in an individual in need thereof comprisingadministering the composition according to claim 44 to the individual inan effective amount such that antibody production contributing to humansystemic lupus erythematosus is suppressed.
 30. A method of making aconjugate according to claim 16, said method comprising the steps of:(a) reacting a plurality of first single-stranded polynucleotides tosaid valency platform for a time and under conditions effective to forma conjugate between the valency platform and the plurality of firstsingle-stranded polynucleotides; (b) annealing a complementarysingle-stranded polynucleotide to said first single-strandedpolynucleotides for a time and under conditions effective to form theconjugate according to claim 66; and (c) isolating the conjugate of step(b).
 31. A conjugate according to claim 13, wherein the valency platformmolecule is derivatized by a reagent selected from the group consistingof 3,5-bis-(iodoacetamido) benzoyl chloride,3-carboxypropionamide-N,N-bis[(6‘-N’-carboxybenzyloxyaminohexyl)acetamide]4″-nitrophenyl ester,3-carboxypropionamide-N,N-bis-[(8‘-N’-carbobenzyloxyamino-3′,6′-dioxaoctyl)acetamide]4″-nitrophenylester, and N,N-di(2-[6‘-N’-carbobenzyloxy-aminohexanoamido]ethyl)amine.32. A conjugate according to claim 16, wherein the polynucleotide duplexis (CA)₁₀: (TG)₁₀ and the valency platform molecule is derivatizedtriethylene glycol.
 33. A conjugate according to claim 32, wherein themolar ratio of duplex to valency platform molecule is in the range of2:1 to 8:1.
 34. A conjugate according to claim 32, wherein the molarratio of duplex to valency platform molecule is 4:1.
 35. A conjugateaccording to claim 16, wherein the polynucleotide duplex is (CA)₁₀:(TG)₁₀ and the valency platform molecule is derivatized2,2′-ethylenedioxydiethylamine.
 36. A conjugate according to claim 35,wherein the molar ratio of duplex to valency platform molecule is in therange of 2:1 to 8:1.
 37. A conjugate according to claim 35, wherein themolar ratio of duplex to valency platform molecule is 4:1.
 38. Aconjugate according to claim 13, wherein the immunogen is an externalimmunogen.
 39. A conjugate according to claim 38, wherein the externalimmunogen is a biological drug, an allergen or a Rhesus (Rh/D) immunogenassociated with Rh hemolytic disease, α-sperm associated with maleinfertility or the carbohydrate complex associated with rheumatic fever.40. A conjugate according to claim 13, wherein the immunogen is aself-immunogen.
 41. A conjugate according to claim 38, wherein theself-immunogen contributes to thyroiditis, diabetes, stroke ormyasthenia gravis.
 42. A conjugate according to claim 13, wherein theimmunogen and the analog are polypeptides.
 43. A composition comprisinga conjugate according to claim 13 formulated with a pharmaceuticallyacceptable carrier.
 44. A composition comprising a conjugate accordingto claim 16 formulated with a pharmaceutically acceptable carrier. 45.The conjugate according to claim 16 wherein said polynucleotide duplexcontains at least 20 base pairs.